Mutant Aequorea victoria fluorescent proteins having increased cellular fluorescence

ABSTRACT

The present invention is directed to mutants of the jellyfish Aequorea victoria green fluorescent protein (GFP) having at least 5 and preferably greater than 20 times the specific green fluorescence of the wild type protein. In other embodiments, the invention comprises mutant blue fluorescent proteins (BFPs) that emit an enhanced blue fluorescence. The invention also encompasses the expression of nucleic acids that encode a mutant GFP or BFP in a wide variety of engineered host cells, and the isolation of engineered proteins having increased fluorescent activity. The novel mutants of the present invention allow for a significantly more sensitive detection of fluorescence in engineered host cells than is possible with GFP or with its known mutants. Thus, the mutant fluorescent proteins provided herein can be used as sensitive reporter molecules to detect the cell and tissue-specific expression and subcellular compartmentalization of GFP or BFP mutants, or of chimeric proteins comprising GFP or BFP mutants fused to a regulatory sequence or to a second protein sequence.

FIELD OF THE INVENTION

This invention generally relates to novel proteins and their productionwhich are useful for detecting gene expression and for visualizing thesubcellular targeting and distribution of selected proteins andpeptides, among other things. The invention specifically relates tomutations in the gene coding for the jellyfish Aequorea victoria greenfluorescent protein ("GFP"), which mutations encode mutant GFP proteinshaving either an enhanced green or a blue fluorescence, and uses forthem.

BACKGROUND OF THE INVENTION

Green fluorescent protein ("GFP") is a monomeric protein of about 27 kDawhich can be isolated from the bioluminescent jellyfish Aequoreavictoria. When wild type GFP is illuminated by blue or ultravioletlight, it emits a brilliant green fluorescence. Similar to fluoresceinisothiocyanate, GFP absorbs ultraviolet and blue light with a maximumabsorbance at 395 nm and a minor peak of absorbance at 470 nm, and emitsgreen light with a maximum emission at 509 nm with a minor peak at 540nm. GFP fluorescence persists even after fixation with formaldehyde, andit is more stable to photobleaching than fluorescein.

The gene for GFP has been isolated and sequenced. Prasher, D. C. et al.(1992), "Primary structure of the Aequorea victoria green fluorescentprotein," Gene 111:229-233. Expression vectors that comprise the GFPgene or cDNA have been introduced into a variety of host cells. Thesehost cells include: Chinese hamster ovary (CHO) cells, human embryonickidney cells (HEK293), COS-1 monkey cells, myeloma cells, NIH 3T3 mousefibroblasts, PtK1 cells, BHK cells, PC12 cells, Xenopus, leech,transgenic zebra fish, transgenic mice, Drosophila and several plants.The GFP molecules expressed by these different cells have a similarfluorescence as the native molecules, demonstrating that the GFPfluorescence does not require any species-specific cofactors orsubstrates. See, e.g., Baulcombe, D. et al. (1995), "Jellyfish greenfluorescent protein as a reporter for virus infections," The PlantJournal 7:1045-1053; Chalfie, M. et al. (1994), "Green fluorescentprotein as a marker for gene expression," Science 263:802-805; Inouye,S. & Tsuji, F. (1994), "Aequorea green fluorescent protein: expressionof the gene and fluorescent characteristics of the recombinant protein,"FEBS Letters 341:277-280; Inouye, S. & Tsuji, F. (1994), "Evidence forredox forms of the Aequorea green fluorescent protein," FEBS Letters351:211-214; Kain, S. et al. (1995), "The green fluorescent protein as areporter of gene expression and protein localization," BioTechniques (inpress); Kitts, P. et al. (1995), "Green Fluorescent Protein (GFP): Anovel reporter for monitoring gene expression in living organisms,"CLONTECHniques X(1):1-3; Lo, D. et al. (1994), "Neuronal transfection inbrain slices using particle-mediated gene transfer," Neuron13:1263-1268; Moss, J. B. & Rosenthal, N. (1994), "Analysis of geneexpression patterns in the embryonic mouse myotome with the greenfluorescent protein, a new vital marker," J. Cell. Biochem., Supplement18D W161; Niedz, R. et al. (1995), "Green fluorescent protein: an invivo reporter of plant gene expression," Plant Cell Reports 14:403-406;Wu, G.-I. et al. (1995), "Infection of frog neurons with vaccinia viruspermits in vivo expression of foreign proteins," Neuron 14:681-684; Yu,J. & van den Engh, G. (1995), "Flow-sort and growth of single bacterialcells transformed with cosmid and plasmid vectors that include the genefor green-fluorescent protein as a visible marker," Abstracts of paperspresented at the 1995 meeting on "Genome Mapping and Sequencing," ColdSpring Harbor, p. 293.

The active GFP chromophore is a hexapeptide which contains a cyclizedSer-dehydroTyr-gly trimer at positions 65-67. This chromophore is onlyfluorescent when embedded within the intact GFP protein. Chromophoreformation occurs post-translationally; nascent GFP is not fluorescent.The chromophore is thought to be formed by a cyclization reaction and anoxidation step that requires molecular oxygen.

Proteins can be fused to the amino (N-) or carboxy (C-) terminus of GFP.Such fused proteins have been shown to retain the fluorescent propertiesof GFP and the functional properties of the fusion partner. Bian, J. etal. (1995), "Nuclear localization of HIV-1 matrix protein P17: The useof A. victoria GFP in protein tagging and tracing," FASEB J. 9:AI279;Flach, J. et al. (1994), "A yeast RNA-binding protein shuttles betweenthe nucleus and the cytoplasm," Mol. Cell. Biol. 14:8399-8407; Marshall,J. et al. (1995), "The jellyfish green fluorescent protein: a new toolfor studying ion channel expression and function," Neuron 14:211-215;Olmsted, J. et al. (1994), "Green Fluorescent Protein (GFP) chimeras asreporters for MAP4 behavior in living cells," Mol. Biol. of the Cell5:167a; Rizzuto, R. et al. (1995), "Chimeric green fluorescent proteinas a tool for visualizing subcellular organelles in living cells,"Current Biol. 5:635-642; Sengupta, P. et al. (1994), "The C. elegansgene odr-7 encodes an olfactory-specific member of the nuclear receptorsuperfamily," Cell 79:971-980; Stearns, T. (1995), "The greenrevolution," Current Biol. 5:262-264; Treinin, M. & Chalfie, M. (1995),"A mutated acetylcholine receptor subunit causes neuronal degenerationin C. elegans," Neuron 14:871-877; Wang, S. & Hazelrigg, T. (1994),"Implications for bcd MRNA localization from spatial distribution of exuprotein in Drosophila oogenesis," Nature 369:400-403.

A number of GFP mutants have been reported. Delagrave, S. et al. (1995)"Red-shifted excitation mutants of the green fluorescent protein,"Bio/Technology 13:151-154; Heim, R. et al. (1994) "Wavelength mutationsand posttranslational autoxidation of green fluorescent protein," Proc.Natl. Acad. Sci. USA 91:12501-12504; Heim, R. et al. (1995), "Improvedgreen fluorescence," Nature 373:663-664. Delgrave et al. (1995)Bio/Technology 13:151-154 isolated mutants of cloned Aequorea victoriaGFP that had red-shifted excitation spectra. Heim, R. et al. (1994)"Wavelength mutations and posttranslational autoxidation of greenfluorescent protein," Proc. Natl. Acad. Sci. USA 91:12501-12504 reporteda mutant (Tyr66 to His) having a blue fluorescence, which is hereindesignated BFP(Tyr₆₇ →His). These references have neither taught norsuggested that their mutations resulted in an increase in the cellularfluorescence of the mutant GFPs.

In general, the level of fluorescence of a protein expressed in a celldepends on several factors, such as number of copies made of thefluorescent protein, stability of the protein, efficiency of formationof the chromophore, and interactions with cellular solvents, solutes andstructures. Although the fluorescent signal from wild type GFP or fromthe reported mutants is generally adequate for bulk detection ofabundantly expressed GFP or of GFP-containing chimeras, it is inadequatefor detecting transient low or constitutively low levels of expression,or for performing fine structural subcellular localizations. Thislimitation severely restricts the use of native GFP or of the reportedmutants as a biochemical and structural marker for gene expression andmorphological studies.

SUMMARY OF THE INVENTION

It an object of the invention to provide engineered GFP-encoding nucleicacid sequences that encode modified GFP molecules having a greatercellular fluorescence than modified wild type GFP having SEQ ID NO:2 orpreviously described recombinant GFP.

It is a further object of this invention to provide recombinant vectorscontaining these modified GFP-encoding nucleic acid sequences, whichvectors are capable of being inserted into a variety of cells (includingmammalian and eukaryotic cells) and expressing the modified GFP.

It is also an object of this invention to provide host cells capable ofproviding useful quantities of homogeneous modified GFP.

It is yet another object of this invention to provide peptides thatpossess a greater cellular fluorescence than native GFP or unalteredrecombinant GFP and that can be produced in large quantities in alaboratory, by a microorganism or by a cell in culture.

These and other objects of the invention have been accomplished byproviding mutant GFP-encoding nucleic acids whose gene product exhibitsan increased cellular fluorescence relative to naturally occurring orrecombinantly produced wild type GFP ("wtGFP"). In some embodiments, themodified GFPs possess fluorescent activity that is 50-100 fold greaterthan that of unmodified GFP.

The modified proteins of the present invention are produced by makingmutations in a genetic sequence that result in alterations in the aminoacid sequence of the resulting gene product. Our starting material was aGFP-encoding nucleic acid wherein a codon encoding an additional nucleicacid was inserted at position 2 of the previously published GFP aminoacid sequence (Chalfie et al., 1994), to introduce a useful restrictionsite. Due to the amino acid insertion at position 2 of the GFP aminoacid sequence, our numbering of the GFP amino acids and description ofthe amino acid amutations is off by one as compared to the originallyreported wild type GFP sequence (Prasher et al., 1992). Thus, amino acid65 by our numbering corresponds to amino acid 64 of the originallyreported wild type GFP, amino acid 168 corresponds to amino acid 167 ofthe originally reported wild type GFP, etc.

Using the modified modified wild type GFP having SEQ ID NO:2 describedherein, a number of the unique mutants described herein derive from thediscovery of an unplanned and unexpected mutation called "SG12",obtained in the course of site-directed mutagenesis experiments, whereina phenylalanine at position 65 of mwtGFP was converted to leucine. Amutant referred to as "SG11," which combined the phenylalanine 65 toleucine alteration with an isoleucine 168 to threonine substitution anda lysine 239 to asparagine substitution, gave a further enhancedfluorescence intensity. The lysine 239 to asparagine substitution doesnot affect the fluorescence of GFP; indeed the C-terminal lysine orasparagine may be deleted without affecting fluorescence. A third andfurther improved GFP mutant was obtained by further mutating "SG11."This mutant is referred to as "SG25" and, in addition to the SG11mutations, contains an additional mutation, a substitution of a cysteineat position 66 for the serine normally found at that position in thesequence.

In addition, the invention encompasses novel GFP mutants that emit ablue fluorescence. These blue mutants are derived from a mutation of thewild type GFP (Heim, R. et al. (1994) "Wavelength mutations andposttranslational autoxidation of green fluorescent protein," Proc.Natl. Acad. Sci. USA 91:12501-12504), in which histidine was substitutedfor tyrosine at amino acid position 66. This mutant emits a bluefluorescence, i.e., it becomes a Blue Fluorescent Protein (BFP).

Novel BFP mutants having an enhanced blue fluorescence were made byfurther modifying this BFP(Tyr₆₇ →His). The introduction of the samemutation used to generate SG12, (i.e., phenylalanine to leucine atposition 65) into BFP(Tyr₆₇ →His) resulted in a new mutant having abrighter fluorescence, designated "SuperBlue-42" (SB42). A secondindependently generated mutation of BFP(Tyr₆₇ →His), in which a valineat position 164 was converted to alanine, also emitted an enhanced bluefluorescent signal and is referred to as "SB49." A combination of theabove two mutations resulted in "SB50", which exhibited an even greaterfluorescence enhancement than either of the previous mutations.

The novel GFP and BFP mutants of this invention allow for asignificantly more sensitive detection of fluorescence in host cellsthan is possible with the wild type protein. Accordingly, the mutantGFPs provided herein can be used, among other things, as sensitivereporter molecules to detect the cell and tissue-specific expression andsubcellular compartmentalization of GFP or of chimeric proteinscomprising GFP fused to a regulatory sequence or to a second proteinsequence. In addition, these mutations make possible a variety of oneand two color protein assays to quantitate expression in mammaliancells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises mutant nucleic acids that encodeengineered GFPs having a greater cellular fluorescence than eithernative GFP or unaltered recombinant GFP, and the mutant GFPs themselves.It further comprises a subset of mutant GFPs that are mutant bluefluorescent proteins ("BFPs") that are derived from a published BFP,designated BFP(Tyr₆₇ →His), wherein the mutant BFPs have a cellularfluorescence that is at least five times greater, preferably ten timesgreater, and most preferably 20 times greater than that of BFP(Tyr₆₇→His). The invention also encompasses compositions such as vectors andcells that comprise either the mutant nucleic acids or the mutantprotein gene products. The mutant GFP nucleic acids and proteins may beused to detect and quantify gene expression in living cells, and todetect and quantify tissue specific expression and subcellulardistribution of GFP or of GFP fused to other proteins.

I. General Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al. (1994)Dictionary of Microbiology and Molecular Biology, second edition, JohnWiley and Sons (New York) provides one of skill with a generaldictionary of many of the terms used in this invention. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are described. For purposes of thepresent invention, the following terms are defined below.

The symbols, abbreviations and definitions used herein are set forthbelow:

DNA, deoxyribonucleic acid

RNA, ribonucleic acid

mRNA, messenger RNA

cDNA, complementary DNA (enzymatically synthesized from an mRNAsequence)

A--Adenine

T--Thymine

G--Guanine

C--Cytosine

U--Uracil

GFP, Green Fluorescent Protein

BFP, Blue Fluorescent Protein

Amino acids are sometimes referred to herein by the conventional one orthree letter codes.

Modified wild type green fluorescent protein ("mwtGFP") refers to the239 amino acid sequence described by Chalfie et al., Science 263,802-805, 1994, the nucleotide sequence of which is set out as SEQ IDNO:1, and the amino acid sequence of which is set out as SEQ ID NO:2.This sequence differs from the original 238 amino acid GFP isolated fromthe bioluminescent jellyfish Aequorea victoria in that one amino acidhas been inserted after position 2 of the 238 amino acid sequence. Whenreference in this application is made to an amino acid position of GFP,the position is made with reference to that described by Chalfie et al.,supra and thus of SEQ ID NO:2.

The term "blue fluorescent protein" (BFP) refers to mutants of wtGFPwherein the tyrosine at position 67 is converted to a histidine, whichmutants emit a blue fluorescence. The non-limiting prototype is hereindesignated BFP(Tyr₆₇ →His).

A shorthand designation for mutations that result in a change in aminoacid sequence is the one or three letter code for the original aminoacid, the number of the position of the amino acid in the mwtGFPsequence, followed by the one or three letter code for the new aminoacid. Thus, Phe65Leu or F65L both designate a mutation wherein thephenylalanine at position 65 of the mwtGFP is converted to leucine.

Salts of any of the proteins described herein will naturally occur whensuch proteins are present in (or isolated from) aqueous solutions ofvarious pHs. All salts of peptides having the indicated biologicalactivity are considered to be within the scope of the present invention.Examples include alkali, alkaline earth, and other metal salts ofcarboxylic acid residues, acid addition salts (e.g., HCl) of aminoresidues, and Zwitterions formed by reactions between carboxylic acidand amino acid residues within the same molecule.

The terms "bioluminescent" and "fluorescent" refer to the ability of GFPor of a derivative thereof to emit light ("emitted or fluorescentlight") of a characteristic wavelength when excited by light which isgenerally of a characteristic and different wavelength than that used togenerate the emission.

The term "cellular fluorescence" denotes the fluorescence of aGFP-derived protein of the present invention when expressed in a cell,especially a mammalian cell.

The term "nucleic acid" refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless specifically limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence implicitly provides the complementarysequence thereof, as well as the sequence explicitly indicated. As usedherein, the terms "nucleic acid" and "gene" are interchangeable, andthey encompass the term "cDNA."

The phrase "a nucleic acid sequence encoding" refers to a nucleic acidwhich contains sequence information that, if translated, yields theprimary amino acid sequence of a specific protein or peptide. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences which may be introduced to conform with codon preference in aspecific host cell.

The phrase "nucleic acid construct" denotes a nucleic acid that iscomposed of two or more nucleic acid sequences that are derived fromdifferent sources and that are ligated together using methods known inthe art.

The term "regulatory sequence" denotes all the non-coding elements of anucleic acid sequence required for the correct and efficient expressionof the "coding region" (i.e., the region that actually encodes the aminoacid sequence of a peptide or protein), e.g., binding cites forpolymerases and transcription factors, transcription and translationinitiation and termination sequences, TATA box, a promoter to directtranscription, a ribosome binding site for translational initiation,polyadenylation sequences, enhancer elements.

The term "isolated" refers to material which is substantially oressentially free from components which normally accompany it as found inits native state (for example, a band on a gel). The isolated nucleicacids and the isolated proteins of this invention do not containmaterials normally associated with their in situ environment, inparticular, nuclear, cytosolic or membrane associated proteins ornucleic acids other than those nucleic acids which are indicated. Theterm "homogeneous" refers to a peptide or DNA sequence where the primarymolecular structure (i.e., the sequence of amino acids or nucleotides)of substantially all molecules present in the composition underconsideration is identical. The term "substantially" used in thepreceding sentences preferably means at least 80% by weight, morepreferably at least 95% by weight, and most preferably at least 99% byweight.

The nucleic acids of this invention, whether RNA, cDNA, genomic DNA, ora hybrid of the various combinations, are synthesized in vitro or areisolated from natural sources or recombinant clones. The nucleic acidsclaimed herein are present in transformed or transfected whole cells, intransformed or transfected cell lysates, or in a partially purified orsubstantially pure form. The nucleic acids of the present invention areobtained as homogeneous preparations. They may be prepared by standardtechniques well known in the art, including selective precipitation withsuch substances as ammonium sulfate, isopropyl alcohol, ethyl alcohol,and/or exclusion, ion exchange or affinity column chromatography,immunopurification methods, and others.

The phrase "conservatively modified variants thereof," when used withreference to a protein, denotes conservative amino acid substitutions inwhich both the original and the substituted amino acids have similarstructure (e.g., the R group contains a carboxylic acid) and properties(e.g., the original and the substituted amino acids are acidic, such asglutamic and aspartic acid), such that the substitutions do notessentially alter specified properties of the protein, such asfluorescence. Amino acid substitutions that are conservative are wellknown in the art. The phrase "conservatively modified variants thereof,"when used to describe a reference nucleic acid, denotes nucleic acidshaving nucleotide substitutions that yield degenerate codons for a givenamino acid or that encode conservative amino acid substitutions, ascompared to the reference nucleic acid.

The term "recombinant" or "engineered" when used with reference to anucleic acid or a protein generally denotes that the composition orprimary sequence of said nucleic acid or protein has been altered fromthe naturally occurring sequence using experimental manipulations wellknown to those skilled in the art. It may also denote that a nucleicacid or protein has been isolated and cloned into a vector, or that thenucleic acid that has been introduced into or expressed in a cell orcellular environment other than the cell or cellular environment inwhich said nucleic acid or protein may be found in nature. The phrase"engineered Aequorea victoria fluorescent protein" specificallyencompasses a protein obtained by introducing one or more sequencealterations into the coding region of a nucleic acid that encodesmodified wild type Aequorea victoria GFP having SEQ ID NO:2, wherein thegene product of the engineered nucleic acid is a fluorescent proteinrecognized by antisera to modified wild type Aequorea Victoria GFP.

The term "recombinant" or "engineered" when used with reference to acell indicates that, as a result of experimental manipulation, the cellreplicates or expresses a nucleic acid or expresses a peptide or proteinencoded by a nucleic acid, whose origin is exogenous to the cell.Recombinant cells can express nucleic acids that are not found withinthe native (non-recombinant) form of the cell. Recombinant cells canalso express nucleic acids found in the native form of the cell whereinthe nucleic acids are re-introduced into the cell by artificial means.

The term "vector" denotes an engineered nucleic acid construct thatcontains sequence elements that mediate the replication of the vectorsequence and/or the expression of coding sequences present on thevector. Examples of vectors include eukaryotic and prokaryotic plasmids,viruses (for example, the HIV virus), cosmids, phagemids, and the like.The term "operably linked" refers to functional linkage between a firstnucleic acid (for example, an expression control sequence such as apromoter or an array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.One or more selected isolated nucleic acids may be operably linked to avector by methods known in the art.

"Transduction" or "transformation" denotes the process whereby exogenousextracellular DNA is introduced into a cell, such that the cell iscapable of replicating and or expressing the exogenous DNA. Generally, aselected nucleic acid is first inserted into a vector and the vector isthen introduced into the cell. For example, plasmid DNA that isintroduced under appropriate environmental conditions may undergoreplication in the transformed cell, and the replicated copies aredistributed to progeny cells when cell division occurs. As a result, anew cell line is established, containing the plasmid and carrying thegenetic determinants thereof. Transformation by a plasmid in thismanner, where the plasmid genes are maintained in the cell line byplasmid replication, occurs at high frequency when the transformingplasmid DNA is in closed loop form, and does not or rarely occurs iflinear plasmid DNA is used.

All the patents and publications cited in this disclosure are indicativeof the level of skill of those skilled in the art to which thisinvention pertains and are all herein individually incorporated byreference for all purposes.

II. The GFP Mutants and Their Expression

A. The GFP mutants

The isolated nucleic acids reported here are those that encode anengineered protein derived from Aequorea victoria green fluorescentprotein ("GFP") having a fluorescence at maximum emission that is atleast five times greater, preferably ten times greater, and mostpreferably twenty times greater than the fluorescence at maximumemission of modified wild type GFP. In one embodiment, a nucleic acidencodes for leucine at amino acid position 65. This amino acid positionis important for the enhanced fluorescence. In another embodiment theengineered isolated GFP nucleic acid also encodes for threonine at aminoacid position 168. In an additional embodiment, the engineered isolatedGFP nucleic acid further encodes for cysteine at amino acid position 66.

Also described here are GFP mutants that have enhanced blue fluorescentproperties. These mutants have an isolated nucleic acid that encode anengineered Aequorea victoria blue fluorescent protein that encodes forhistidine at amino acid position 67, leucine at amino acid position 65and has a cellular fluorescence that is at least five times greater,preferably 10 times greater, most preferably 20 times greater than thatof BFP(Tyr₆₇ →His). An alternative isolated BFP nucleic acid is one thatencodes for an engineered Aequorea victoria blue fluorescent proteinwherein the engineered BFP has histidine at amino acid position 67 andalanine at amino acid position 164. A third engineered isolated BFPnucleic acid sequence is one that has histidine at amino acid position67, leucine at amino acid position 65 and alanine at amino acid position164.

The nucleic acid and amino acid sequences for the modified wild type GFPare set out in SEQ ID NO:1 and SEQ ID NO:2. The sequence is well-known,well-described and readily available for manipulation and use. Vectorsbearing the nucleic acid sequence are commercially readily availablefrom, for example, Clontech Laboratories, Inc., Clontech Laboratories,Inc., Palo Alto, Calif. Clontech provides a line of reporter vectors forGFP, including the cDNA construct described by Chalfie, et al., supra, apromoterless GFP vector for monitoring the expression of clonedpromoters in mammalian cells, and a series of vectors for creatingfusion proteins to either the amino or carboxy terminus of GFP.

One of skill in the art will recognize many ways of generatingalterations in a given nucleic acid sequence. Such well-known methodsinclude site-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology Volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, (Sambrook); and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel); Pirrung et al., U.S. Pat. No. 5,143,854; andFodor et al., Science, 251, 767-77 (1991). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide information useful in known biological methods. Suchmanufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&Dsystems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill. Using these techniques, it ispossible to substitute at will any nucleotide in a nucleic acid thatencodes any GFP or BFP disclosed herein or any amino acid in a GFP orBFP described herein for a predetermined nucleotide or amino acid. Forexample, it is possible to generate at will modified GFPs and BFP(Tyr₆₇→His)s that contain leucine at position 65 and one or two or threeadditional mutations at any other position of the mwtGFP or BFP(Tyr₆₇→His).

The sequence of the cloned genes and synthetic oligonucleotides can beverified using the chemical degradation method of A. M. Maxam et al.(1980), Methods in Enzymology 65:499-560. The sequence can be confirmedafter the assembly of the oligonucleotide fragments into thedouble-stranded DNA sequence using the method of Maxam and Gilbert,supra, or the chain termination method for sequencing double-strandedtemplates of R. B. Wallace et al. (1981), Gene, 16:21-26. DNA sequencingmay also be performed by the PCR-assisted fluorescent terminator method(ReadyReaction DyeDeoxy Terminator Cycle Sequencing Kit, ABI, Columbia,Md.) according to the manufacturer's instructions, using the ABI Model373A DNA Sequencing System. Sequencing data is analyzed using thecommercially available Sequencher program (Gene Codes, Gene Codes, AnnArbor, Mich.).

B. Expression of Mutant GFP

Clearly, the nucleic acid sequences of the present invention areexcellent reporter sequences since the expressed proteins can be readilydetected by fluorescence as described below. The sequences can be usedin conjunction with any application appreciated to date for GFP andfurther in applications where a greater degree of fluorescence isrequired. Expression of the sequences described herein whetherexpression is desired alone or in combination with other sequences ofinterest is described below.

Vectors to which selected foreign nucleic acids are operably linked maybe used to introduce these selected nucleic acids into host cells andmediate their replication and/or expression. Cloning vectors are usefulfor replicating the foreign nucleic acids and obtaining clones ofspecific foreign nucleic acid-containing vectors. Expression vectorsmediate the expression of the foreign nucleic acid. Some vectors areboth cloning and expression vectors.

Once a nucleic acid is synthesized or isolated and inserted into avector and cloned, one may express the nucleic acid in a variety ofrecombinantly engineered cells known to those of skill in the art. Asused herein, "expression" refers to transcription of nucleic acids,either without or preferably with subsequent translation.

Expression of a mutant BFP or of modified wild type or mutant GFP can beenhanced by including multiple copies of the GFP-encoding nucleic acidin a transformed host, by selecting a vector known to reproduce in thehost, thereby producing large quantities of protein from exogenousinserted DNA (such as pUC8, ptac12, or pIN-III-ompA1, 2, or 3), or byany other known means of enhancing peptide expression. In all cases,mwtGFP or mutant GFPs will be expressed when the DNA sequence isfunctionally inserted into a vector. "Functionally inserted" means thatit is inserted in proper reading frame and orientation. Typically, a GFPgene will be inserted downstream from a promoter and will be followed bya stop codon, although production as a hybrid protein followed bycleavage may be used, if desired.

Examples of cells which are suitable for the cloning and expression ofthe nucleic acids of the invention include bacteria, yeast, filamentousfungi, insect (especially employing baculoviral vectors), and mammaliancells, in particular cells capable of being maintained in tissueculture.

Host cells are competent or rendered competent for transformation byvarious means. There are several well-known methods of introducing DNAinto animal cells. These include: calcium phosphate precipitation,fusion of the recipient cells with bacterial protoplasts containing theDNA, treatment of the recipient cells with liposomes containing the DNA,DEAE dextran, receptor-mediated endocytosis, electroporation andmicro-injection of the DNA directly into the cells.

It is expected that those of skill in the art are knowledgeable in thenumerous systems available for cloning and expression of nucleic acids.In brief summary, the expression of natural or synthetic nucleic acidsis typically achieved by operably linking a nucleic acid of interest toa promoter (which is either constitutive or inducible), andincorporating the construct into an expression vector. The vectors aresuitable for replication and integration in prokaryotes, eukaryotes, orboth. Typical cloning vectors contain transcription and translationterminators, transcription and translation initiation sequences, andpromoters useful for regulation of the expression of the particularnucleic acid. The vectors optionally comprise generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in eukaryotes, orprokaryotes, or both, (e.g., shuttle vectors) and selection markers forboth prokaryotic and eukaryotic systems. See, e.g., Sambrook and Ausbel(both supra).

1. Expression in Prokaryotes

Prokaryotic systems for cloning and/or expressing engineered GFP or BFPproteins are available using E. coli, Bacillus sp. and Salmonella(Palva, I. et al. (1983), Gene 22:229-235; Mosbach, K. et al. (1983),Nature 302:543-545. To obtain high level expression in a prokaryoticsystem of a cloned nucleic acid such as those encoding engineered GFPsor BFPs, it is essential to construct expression vectors which contain,at a minimum, a strong promoter to direct transcription, a ribosomebinding site for translational initiation, a transcription/translationterminator, a bacterial replicon, a nucleic acid encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of foreign nucleic acids. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. Examples of regulatoryregions suitable for this purpose in E. coli are the promoter andoperator region of the E. coli tryptophan biosynthetic pathway asdescribed by Yanofsky, C. (1984), J. Bacteriol., 158:1018-1024, and theleftward promoter of phage lambda (P_(L)) as described by Herskowitz, I.and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445 (1980).

The particular vector used to transport the genetic information into thecell is not particularly critical. Any of the conventional vectors usedfor replication, cloning and/or expression in prokaryotic cells may beused.

The foreign nucleic acid can be incorporated into a nonessential regionof the host cell's chromosome. This is achieved by first inserting thenucleic acid into a vector such that it is flanked by regions of DNAhomologous to the insertion site in the host chromosome. Afterintroduction of the vector into a host cell, the foreign nucleic acid isincorporated into the chromosome by homologous recombination between theflanking sequences and chromosomal DNA.

Detection of the expressed protein is achieved by methods known in theart as radioimmunoassays, or Western blotting techniques orimmunoprecipitation. Purification from E. coli can be achieved followingprocedures described in U.S. Pat. No. 4,511,503.

2. Expression in Eukaryotes

Standard eukaryotic transfection methods are used to produce mammalian,yeast or insect cell lines which express large quantities of engineeredGFP or BFP protein which are then purified using standard techniques.See, e.g., Colley et al. (1989), J. Biol. Chem. 264:17619-17622, andGuide to Protein Purification, in Vol. 182 of Methods in Enzymology(Deutscher ed., 1990), D. A. Morrison (1977), J. Bact., 132:349-351, orby J. E. Clark-Curtiss and R. Curtiss (1983), Methods in Enzymology101:347-362, Eds. R. Wu et al., Academic Press, New York.

The particular eukaryotic expression vector used to transport thegenetic information into the cell is not particularly critical. Any ofthe conventional vectors used for expression in eukaryotic cells may beused. Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses are typically used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Barr virus include pHEBO,and p205. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

The expression vector typically comprises a eukaryotic transcriptionunit or expression cassette that contains all the elements required forthe expression of the engineered GFP or BFP DNA in eukaryotic cells. Atypical expression cassette contains a promoter operably linked to theDNA sequence encoding a engineered GFP or BFP protein and signalsrequired for efficient polyadenylation of the transcript.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus, the longterm repeat from various retroviruses such as murine leukemia virus,murine or Rous sarcoma virus and HIV. See, Enhancers and EukaryoticExpression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983,which is incorporated herein by reference.

In the construction of the expression cassette, the promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

If the mRNA encoded by the structural gene is to be efficientlytranslated, polyadenylation sequences are also commonly added to thevector construct. Two distinct sequence elements are required foraccurate and efficient polyadenylation: GU or U rich sequences locateddownstream from the polyadenylation site and a highly conserved sequenceof six nucleotides, AAUAAA, located 11-30 nucleotides upstream.Termination and polyadenylation signals that are suitable for thepresent invention include those derived from SV40, or a partial genomiccopy of a gene already resident on the expression vector.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the transfectedDNA. For instance, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The DNA sequence encoding the engineered GFP or BFP protein maytypically be linked to a cleavable signal peptide sequence to promotesecretion of the encoded protein by the transformed cell. Such signalpeptides would include, among others, the signal peptides from tissueplasminogen activator, insulin, neuron growth factor, and juvenilehormone esterase of Heliothis virescens. Additional elements of thecassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

The vector may or may not comprise a eukaryotic replicon. If aeukaryotic replicon is present, then the vector is amplifiable ineukaryotic cells using the appropriate selectable marker. If the vectordoes not comprise a eukaryotic replicon, no episomal amplification ispossible. Instead, the transfected DNA integrates into the genome of thetransfected cell, where the promoter directs expression of the desirednucleic acid.

The vectors usually comprise selectable markers which result in nucleicacid amplification such as the sodium, potassium ATPase, thymidinekinase, aminoglycoside phosphotransferase, hygromycin Bphosphotransferase, xanthine-guanine phosphoribosyl transferase, CAD(carbamyl phosphate synthetase, aspartate transcarbamylase, anddihydroorotase), adenosine deaminase, dihydrofolate reductase, andasparagine synthetase and ouabain selection. Alternatively, high yieldexpression systems not involving nucleic acid amplification are alsosuitable, such as using a bacculovirus vector in insect cells, with theengineered GFP or BFP encoding sequence under the direction of thepolyhedrin promoter or other strong baculovirus promoters.

The expression vectors of the present invention will typically containboth prokaryotic sequences that facilitate the cloning of the vector inbacteria as well as one or more eukaryotic transcription units that areexpressed only in eukaryotic cells, such as mammalian cells. Theprokaryotic sequences are preferably chosen such that they do notinterfere with the replication of the DNA in eukaryotic cells.

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign nucleic acidic material into a host cell(see Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure utilized be capable of successfullyintroducing at least one nucleic acid into the host cell which iscapable of expressing the engineered GFP or BFP protein.

3. Expression in insect cells

The baculovirus expression vector utilizes the highly expressed andregulated Autographa californica nuclear polyhedrosis virus (AcMNPV)polyhedrin promoter modified for the insertion of foreign nucleic acids.Synthesis of polyhedrin protein results in the formation of occlusionbodies in the infected insect cell. The baculovirus vector utilizes manyof the protein modification, processing, and transport systems thatoccur in higher eukaryotic cells. The recombinant eukaryotic proteinsexpressed using this vector have been found in many cases to be,antigenically, immunogenically, and functionally similar to theirnatural counterparts.

Briefly, a DNA sequence encoding an engineered GFP or BFP is insertedinto a transfer plasmid vector in the proper orientation downstream fromthe polyhedrin promoter, and flanked on both ends with baculovirussequences. Cultured insect cells, commonly Spodoptera frugiperda cells,are transfected with a mixture of viral and plasmid DNAs. The virus thatdevelop, some of which are recombinant virus that result from homologousrecombination between the two DNAs, are plated at 100-1000 plaques perplate. The plaques containing recombinant virus can be identifiedvisually because of their ability to form occlusion bodies or by DNAhybridization. The recombinant virus is isolated by plague purification.The resulting recombinant virus, capable of expressing engineered GFP orBFP, is self-propagating in that no helper virus is required formaintenance or replication. After infecting an insect culture withrecombinant virus, one can expect to find recombinant protein within48-72 hours. The infection is essentially lytic within 4-5 days.

There are a variety of transfer vectors into which the engineered GFP orBFP nucleic acid can be inserted. For a summary of transfer vectors seeLuckow, V. A. and Summers, M. D. (1988), Bio/Technology 6:47-55.Preferred is the transfer vector pAcUW21 described by Bishop, D. H. L.(1992) in Seminars in Virology 3:253-264.

4. Retroviral Vectors

Retroviral vectors are particularly useful for modifying eukaryoticcells because of the high efficiency with which the retroviral vectorstransduce target cells and integrate into the target cell genome.Additionally, the retroviruses harboring the retoviral vector arecapable of infecting cells from a wide variety of tissues.

Retroviral vectors are produced by genetically manipulatingretroviruses. Retroviruses are RNA viruses because the viral genome isRNA. Upon infection, this genomic RNA is reverse transcribed into a DNAcopy which is integrated into the chromosomal DNA of transduced cellswith a high degree of stability and efficiency. The integrated DNA copyis referred to as a provirus and is inherited by daughter cells as isany other gene. The wild type retroviral genome and the proviral DNAhave three genes: the gag, the pol and the env genes, which are flankedby two long terminal repeat (LTR) sequences. The gag gene encodes theinternal structural (nucleocapsid) proteins; the pol gene encodes theRNA directed DNA polymerase (reverse transcriptase); and the env geneencodes viral envelope glycoproteins. The 5' and 3' LTRs serve topromote transcription and polyadenylation of virion RNAs. Adjacent tothe 5' LTR are sequences necessary for reverse transcription of thegenome (the tRNA primer binding site) and for efficient encapsulation ofviral RNA into particles (the Psi site). See Mulligan, R. C. (1983), In:Experimental Manipulation of Gene Expression, M. Inouye (ed), 155-173;Mann, R. et al. (1983), Cell, 33:153-159; Cone, R. D. and R. C. Mulligan(1984), Proceedings of the National Academy of Sciences, U.S.A.81:6349-6353.

The design of retroviral vectors is well known to one of skill in theart. See Singer, M. and Berg, P. supra. In brief, if the sequencesnecessary for encapsidation (or packaging of retroviral RNA intoinfectious virions) are missing from the viral genome, the result is acis acting defect which prevents encapsidation of genomic RNA. However,the resulting mutant is still capable of directing the synthesis of allvirion proteins. Retroviral genomes from which these sequences have beendeleted, as well as cell lines containing the mutant genome stablyintegrated into the chromosome are well known in the art and are used toconstruct retroviral vectors. Preparation of retroviral vectors andtheir uses are described in many publications including European PatentApplication EPA 0 178 220, U.S. Pat. No. 4,405,712, Gilboa (1986),Biotechniques 4:504-512, Mann, et al. (1983), Cell 33:153-159, Cone andMulligan (1984), Proc. Natl. Acad. Sci. USA 81:6349-6353, Eglitis, M. A,et al. (1988) Biotechniques 6:608-614, Miller, A. D. et al. (1989)Biotechniques 7:981-990, Miller, A. D. (1992) Nature, supra, Mulligan,R. C. (1993), supra. and Gould, B. et al., and International PatentApplication No. WO 92/07943 entitled "Retroviral Vectors Useful in GeneTherapy." The teachings of these patents and publications areincorporated herein by reference.

The retroviral vector particles are prepared by recombinantly insertingthe nucleic acid encoding engineered GFP or BFP into a retrovirus vectorand packaging the vector with retroviral capsid proteins by use of apackaging cell line. The resultant retroviral vector particle isincapable of replication in the host cell and is capable of integratinginto the host cell genome as a proviral sequence containing theengineered GFP or BFP nucleic acid. As a result, the patient is capableof producing engineered GFP or BFP and metabolize glycogen tocompletion.

Packaging cell lines are used to prepare the retroviral vectorparticles. A packaging cell line is a genetically constructed mammaliantissue culture cell line that produces the necessary viral structuralproteins required for packaging, but which is incapable of producinginfectious virions. Retroviral vectors, on the other hand, lack thestructural genes but have the nucleic acid sequences necessary forpackaging. To prepare a packaging cell line, an infectious clone of adesired retrovirus, in which the packaging site has been deleted, isconstructed. Cells comprising this construct will express all structuralproteins but the introduced DNA will be incapable of being packaged.Alternatively, packaging cell lines can be produced by transforming acell line with one or more expression plasmids encoding the appropriatecore and envelope proteins. In these cells, the gag, pol, and env genescan be derived from the same or different retroviruses.

A number of packaging cell lines suitable for the present invention areavailable in the prior art. Examples of these cell lines include Crip,GPE86, PA317 and PG13. See Miller et al. (1991), J. Virol. 65:2220-2224,which is incorporated herein by reference. Examples of other packagingcell lines are described in Cone, R. and Mulligan, R. C. (1984),Proceedings of the National Academy of Sciences, U.S.A., 81:6349-6353and in Danos, O. and R. C. Mulligan (1988), Proceedings of the NationalAcademy of Sciences, U.S.A., 85:6460-6464, Eglitis, M. A, et al. (1988)Biotechniques 6:608-614, also all incorporated herein by reference.

Packaging cell lines capable of producing retroviral vector particleswith chimeric envelope proteins may be used. Alternatively, amphotropicor xenotropic envelope proteins, such as those produced by PA317 and GPXpackaging cell lines may be used to package the retroviral vectors.

Transforming cells with nucleic acids can involve, for example,incubating the cells with viral vectors (e.g., retroviral oradeno-associated viral vectors) containing with cells within the hostrange of the vector. See, e.g., Methods in Enzymology, Vol. 185,Academic Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) orM. Krieger (1990), Gene Transfer and Expression--A Laboratory Manual,Stockton Press, New York, N.Y., and the references cited therein.

5. Transformation with adeno-associated virus

Adeno associated viruses (AAVs) require helper viruses such asadenovirus or herpes virus to achieve productive infection. In theabsence of helper virus functions, AAV integrates (site-specifically)into a host cell's genome, but the integrated AAV genome has nopathogenic effect. The integration step allows the AAV genome to remaingenetically intact until the host is exposed to the appropriateenvironmental conditions (e.g., a lytic helper virus), whereupon itre-enters the lytic life-cycle. Samulski (1993), Current Opinion inGenetic and Development 3:74-80 and the references cited thereinprovides an overview of the AAV life cycle.

AAV-based vectors are used to transduce cells with target nucleic acids,e.g., in the in vitro production of nucleic acids and peptides, and inin vivo and ex vivo gene therapy procedures. See, West et al. (1987),Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carteret al. (1993), WO 93/24641; Kotin (1994), Human Gene Therapy 5:793-801;Muzyczka (1994), J. Clin. Invest. 94:1351 and Samulski (supra) for anoverview of AAV vectors.

Recombinant AAV vectors (rAAV vectors) deliver foreign nucleic acids toa wide range of mammalian cells (Hermonat & Muzycka (1984), Proc. Natl.Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985), Mol. Cell Biol.5:3251-3260), integrate into the host chromosome (Mclaughlin et al.(1988), J. Virol. 62:1963-1973), and show stable expression of thetransgene in cell and animal models (Flotte et al. (1993), Proc. Natl.Acad. Sci. USA 90:10613-10617). Moreover, unlike some retroviralvectors, rAAV vectors are able to infect non-dividing cells (Podsakoffet al. (1994), J. Virol. 68:5656-66; Flotte et al. (1994), Am. J.Respir. Cell Mol. Biol. 11:517-521). Further advantages of rAAV vectorsinclude the lack of an intrinsic strong promoter, thus avoiding possibleactivation of downstream cellular sequences, and their nakedeicosahedral capsid structure, which renders them stable and easy toconcentrate by common laboratory techniques. rAAV vectors are used toinhibit, e.g., viral infection, by including anti-viral transcriptioncassettes in the rAAV vector which comprise an inhibitor of theinvention.

6. Expression in recombinant vaccinia virus-infected cells

The nucleic acid encoding engineered GFP or BFP is inserted into aplasmid designed for producing recombinant vaccinia, such as pGS62,Langford, C. L. et al. (1986), Mol. Cell. Biol. 6:3191-3199. Thisplasmid consists of a cloning site for insertion of foreign nucleicacids, the P7.5 promoter of vaccinia to direct synthesis of the insertednucleic acid, and the vaccinia TK gene flanking both ends of the foreignnucleic acid.

When the plasmid containing the engineered GFP or BFP nucleic acid isconstructed, the nucleic acid can be transferred to vaccinia virus byhomologous recombination in the infected cell. To achieve this, suitablerecipient cells are transfected with the recombinant plasmid by standardcalcium phosphate precipitation techniques into cells already infectedwith the desirable strain of vaccinia virus, such as Wyeth, Lister, WRor Copenhagen. Homologous recombination occurs between the TK gene inthe virus and the flanking TK gene sequences in the plasmid. Thisresults in a recombinant virus with the foreign nucleic acid insertedinto the viral TK gene, thus rendering the TK gene inactive. Cellscontaining recombinant viruses are selected by adding medium containing5-bromodeoxyuridine, which is lethal for cells expressing a TK gene.

Confirmation of production of recombinant virus is achieved by DNAhybridization using cDNA encoding the engineered GFP or BFP and byimmunodetection techniques using antibodies specific for the expressedprotein. Virus stocks may be prepared by infection of cells such as HeLAS3 spinner cells and harvesting of virus progeny.

7. Expression in cell cultures

GFP- or BFP-encoding nucleic acids can be ligated to various expressionvectors for use in transforming host cell cultures. The culture of cellsused in conjunction with the present invention is well known in the art.Freshney (1994) (Culture of Animal Cells, a Manual of Basic Technique,third edition Wiley-Liss, New York), Kuchler et al. (1977) BiochemicalMethods in Cell Culture and Virology, Kuchler, R. J., Dowden, Hutchinsonand Ross, Inc., and the references cited therein provides a generalguide to the culture of cells. Illustrative cell cultures useful for theproduction of recombinant proteins include cells of insect or mammalianorigin. Mammalian cell systems often will be in the form of monolayersof cells, although mammalian cell suspensions are also used.Illustrative examples of mammalian cell lines include monocytes,lymphocytes, macrophage, VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines (see, e.g.,Freshney, supra).

Cells of mammalian origin are illustrative of cell cultures useful forthe production of the engineered GFP or BFP . Mammalian cell systemsoften will be in the form of monolayers of cells although mammalian cellsuspensions may also be used. Illustrative examples of mammalian celllines include VERO and HeLa cells, Chinese hamster ovary (CHO) celllines, WI38, BHK, COS-7 or MDCK cell lines.

As indicated above, the vector, e.g., a plasmid, which is used totransform the host cell, preferably contains DNA sequences to initiatetranscription and sequences to control the translation of the engineeredGFP or BFP nucleic acid sequence. These sequences are referred to asexpression control sequences. Illustrative expression control sequencesare obtained from the SV-40 promoter (Science 222:524-527, (1983)), theCMV i.e. Promoter (Proc. Natl. Acad. Sci. 81:659-663, (1984)) or themetallothionein promoter (Nature 296:39-42, (1982)). The cloning vectorcontaining the expression control sequences is cleaved using restrictionenzymes and adjusted in size as necessary or desirable and ligated withsequences encoding the engineered GFP or BFP protein by means well knownin the art.

The vectors for transforming cells in culture typically contain genesequences to initiate transcription and translation of the engineeredGFP or BFP gene. These sequences need to be compatible with the selectedhost cell. In addition, the vectors preferably contain a marker toprovide a phenotypic trait for selection of transformed host cells suchas dihydrofolate reductase or metallothionein. Additionally, a vectormight contain a replicative origin.

As mentioned above, when higher animal host cells are employed,polyadenlyation or transcription terminator sequences from knownmammalian genes need to be incorporated into the vector. An example of aterminator sequence is the polyadenylation sequence from the bovinegrowth hormone gene. Sequences for accurate splicing of the transcriptmay also be included. An example of a splicing sequence is the VP1intron from SV40 (Sprague, J. et al. (1983), J. Virol. 45: 773-781).

Additionally gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus type-vectors. Saveria-Campo, M. (1985), "Bovine Papilloma virusDNA a Eukaryotic Cloning Vector" in DNA Cloning Vol. II a PracticalApproach Ed. D. M. Glover, IRL Press, Arlington, Va. pp. 213-238.

The transformed cells are cultured by means well known in the art. Forexample, as published in Kuchler, R. J. et al., (1977), BiochemicalMethods in Cell Culture and Virology.

In addition to the above general procedures which can be used forpreparing recombinant DNA molecules and transformed unicellularorganisms in accordance with the practices of this invention, otherknown techniques and modifications thereof can be used in carrying outthe practice of the invention. Any known system for expression ofisolated genes is suitable for use in the present invention. Forexample, viral expression systems such as the bacculovirus expressionsystem are specifically contemplated within the scope of the invention.Many recent U.S. patents disclose plasmids, genetically engineeringmicroorganisms, and methods of conducting genetic engineering which canbe used in the practice of the present invention. For example, U.S. Pat.No. 4,273,875 discloses a plasmid and a process of isolating the same.U.S. Pat. No. 4,304,863 discloses a process for producing bacteria bygenetic engineering in which a hybrid plasmid is constructed and used totransform a bacterial host. U.S. Pat. No. 4,419,450 discloses a plasmiduseful as a cloning vehicle in recombinant DNA work. U.S. Pat. No.4,362,867 discloses recombinant cDNA construction methods and hybridnucleotides produced thereby which are useful in cloning processes. U.S.Pat. No. 4,403,036 discloses genetic reagents for generating plasmidscontaining multiple copies of DNA segments. U.S. Pat. No. 4,363,877discloses recombinant DNA transfer vectors. U.S. Pat. No. 4,356,270discloses a recombinant DNA cloning vehicle and is a particularly usefuldisclosure for those with limited experience in the area of geneticengineering since it defines many of the terms used in geneticengineering and the basic processes used therein. U.S. Pat. No.4,336,336 discloses a fused gene and a method of making the same. U.S.Pat. No. 4,319,629 discloses plasmid vectors and the production and usethereof. U.S. Pat. No. 4,332,901 discloses a cloning vector useful inrecombinant DNA. Although some of these patents are directed to theproduction of a particular gene product that is not within the scope ofthe present invention, the procedures described therein can easily bemodified to the practice of the invention described in thisspecification by those skilled in the art of genetic engineering.Transferring the isolated GFP cDNA to other expression vectors willproduce constructs which improve the expression of the GFP polypeptidein E. coli or express GFP in other hosts.

III. Detection of GFP and BFP Nucleic Acids and Proteins

A. General detection methods

The nucleic acids and proteins of the invention are detected, confirmedand quantified by any of a number of means well known to those of skillin the art. The unique quality of the inventive expressed proteins hereis that they provide an enhanced fluorescence which can be readily andeasily observed. Fluorescence assays for the expressed proteins aredescribed in detail below. Other general methods for detecting bothnucleic acids and corresponding proteins include analytic biochemicalmethods such as spectrophotometry, radiography, electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,and the like, and various immunological methods such as fluid or gelprecipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.The detection of nucleic acids proceeds by well known methods such asSouthern analysis, northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.

A variety of methods of specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art.For example, one method for evaluating the presence or absence ofengineered GFP or BFP DNA in a sample involves a Southern transfer.Southern et al. (1975), J. Mol. Biol. 98:503. Briefly, the digestedgenomic DNA is run on agarose slab gels in buffer and transferred tomembranes. Hybridization is carried out using the probes discussedabove. Visualization of the hybridized portions allows the qualitativedetermination of the presence or absence of engineered GFP or BFP genes.

Similarly, a Northern transfer may be used for the detection ofengineered GFP or BFP mRNA in samples of RNA from cells expressing theengineered GFP or BFP gene. In brief, the mRNA is isolated from a givencell sample using an acid guanidinium-phenol-chloroform extractionmethod. The mRNA is then electrophoresed to separate the mRNA speciesand the mRNA is transferred from the gel to a nitrocellulose membrane.As with the Southern blots, labeled probes are used to identify thepresence or absence of the engineered GFP or BFP transcript.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in "Nucleic Acid Hybridization, A PracticalApproach," Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985; Galland Pardue (1969), Proc. Natl. Acad. Sci. USA 63:378-383; and John,Burnsteil and Jones (1969), Nature 223:582-587.

For example, sandwich assays are commercially useful hybridizationassays for detecting or isolating nucleic acid sequences. Such assaysutilize a "capture" nucleic acid covalently immobilized to a solidsupport and labelled "signal" nucleic acid in solution. The clinicalsample will provide the target nucleic acid. The "capture" nucleic acidand "signal" nucleic acid probe hybridize with the target nucleic acidto form a "sandwich" hybridization complex. To be effective, the signalnucleic acid cannot hybridize with the capture nucleic acid.

The nucleic acid sequences used in this invention can be either positiveor negative probes. Positive probes bind to their targets and thepresence of duplex formation is evidence of the presence of the target.Negative probes fail to bind to the suspect target and the absence ofduplex formation is evidence of the presence of the target. For example,the use of a wild type specific nucleic acid probe or PCR primers mayact as a negative probe in an assay sample where only the mutantengineered GFP or BFP is present.

Labelled signal nucleic acids, whether those described herein or othersknown in the art are used to detect hybridization. Complementary nucleicacids or signal nucleic acids may be labelled by any one of severalmethods typically used to detect the presence of hybridizedpolynucleotides. One common method of detection is the use ofautoradiography with ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P-labelled probes orthe like. Other labels include ligands which bind to labelledantibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabelled ligand.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label. (Tijssen, P.(1985), "Practice and Theory of Enzyme Immunoassays," LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon, R. H., vanKnippenberg, P. H., Eds., Elsevier, pp. 9-20.)

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR) theligase chain reaction (LCR), Qβ-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA) are found in Berger,Sambrook, and Ausubel, as well as Mullis et al. (1987), U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim& Levinson (Oct. 1, 1990), Chem. Eng. News 36-47; J. NIH Res. (1991)3:81-94; (Kwoh et al. (1989), Proc. Natl. Acad. Sci. USA 86:1173;Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA 87:1874; Lomell etal. (1989), J. Clin. Chem. 35:1826; Landegren et al. (1988), Science241:1077-1080; Van Brunt (1990), Biotechnology 8:291-294; Wu and Wallace(1989), Gene 4:560; Barringer et al. (1990), Gene 89:117, and Sooknananand Malek (1995), Biotechnology 13:563-564. Improved methods of cloningin vitro amplified nucleic acids are described in Wallace et al., U.S.Pat. No. 5,426,039. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBA™, Cangene, Mississauga,Ontario) and Q Beta Replicase systems. These systems can be used todirectly identify mutants where the PCR or LCR primers are designed tobe extended or ligated only when a select sequence is present.Alternatively, the select sequences can be generally amplified using,for example, nonspecific PCR primers and the amplified target regionlater probed for a specific sequence indicative of a mutation.

Oligonucleotides for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981), Tetrahedron Letts. 22(20):1859-1862, e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al. (1984), NucleicAcids Res. 12:6159-6168. Purification of oligonucleotides, wherenecessary, is typically performed by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier (1983), J. Chrom. 255:137-149. The sequence of the syntheticoligonucleotides can be verified using the chemical degradation methodof Maxam and Gilbert (1980) in Grossman and Moldave (eds.) AcademicPress, New York, Methods in Enzymology 65:499-560.

An alternative means for determining the level of expression of theengineered GFP or BFP gene is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer et al. (1987), Methods Enzymol. 152:649-660. In an in situhybridization assay cells are fixed to a solid support, typically aglass slide. If DNA is to be probed, the cells are denatured with heator alkali. The cells are then contacted with a hybridization solution ata moderate temperature to permit annealing of engineered GFP or BFPspecific probes that are labelled. The probes are preferably labelledwith radioisotopes or fluorescent reporters.

B. Fluorescence Assay

When a fluorophore such as protein that is capable of fluorescing isexposed to a light of appropriate wavelength, it will absorb and storelight and then release the stored light energy. The range of wavelengthsthat a fluorophore is capable of absorbing is the excitation spectrumand the range of wavelengths of light that a fluorophore is capable ofemitting is the emission or fluorescence spectrum. The excitation andfluorescence spectra for a given fluorophore usually differ and may bereadily measured using known instruments and methods. For example,scintillation counters and photometers (e.g. luminometers), photographicfilm, and solid state devices such as charge coupled devices, may beused to detect and measure the emission of light.

The nucleic acids, vectors, mutant proteins provided herein, incombination with well known techniques for over-expressing recombinantproteins, make it possible to obtain unlimited supplies of homogeneousmutant GFPs and BFPs. These modified GFPs or BFPs having increasedfluorescent activity replace wtGTP or other currently employed tracersin existing diagnostic and assay systems. Such currently employedtracers include radioactive atoms or molecules and color-producingenzymes such as horseradish peroxidase.

The benefits of using the mutants of the present invention are at leastfour-fold: the modified GFPs and BFPs are safer than radioactive-basedassays, modified GFPs and BFPs can be assayed quickly and easily, andlarge numbers of samples can be handled simultaneously, reducing overallhandling and increasing efficiency. Of great significance, theexpression and subcellular distribution of the fluorescent proteinswithin cells can be detected in living tissues without any otherexperimental manipulation than to placing the cells on a slide andviewing them through a fluorescence microscope. This represents a vastimprovement over methods of immunodetection that require fixation andsubsequent labelling.

The modified GFPs and BFPs of the present invention can be used instandard assays involving a fluorescent marker. For example,ligand-ligator binding pairs that can be modified with the mutants ofthe present invention without disrupting the ability of each to bind tothe other can form the basis of an assay encompassed by the presentinvention. These and other assays are known in the art and their usewith the GFPs and BFPs of the present invention will become obvious toone skilled in the art in light of the teachings disclosed herein.Examples of such assays include competitive assays wherein labeled andunlabeled ligands competitively bind to a ligator, noncompetitive assaywhere a ligand is captured by a ligator and either measured directly or"sandwiched" with a secondary ligator that is labeled. Still other typesof assays include immunoassays, single-step homogeneous assays,multiple-step heterogeneous assays, and enzyme assays.

In a number of embodiments, the mutant GFPs and BFPs are combined withfluorescent microscopy using known techniques (see, e.g., Stauber etal., Virol. 213:439-454 (1995)) or preferably with fluorescenceactivated cell sorting (FACS) to detect and optionally purify or clonecells that express specific recombinant constructs. For a brief overviewof the FACS and its uses, see: Herzenberg et al., 1976, "Fluorescenceactivated cell sorting", Sci. Amer. 234, 108; see also FLOW CYTOMETRYAND SORTING, eds. Melamad, Mullaney and Mendelsohn, John Wiley and Sons,Inc., New York, 1979). Briefly, fluorescence activated cell sorters takea suspension of cells and pass them single file into the light path of alaser placed near a detector. The laser usually has a set wavelength.The detector measures the fluorescent emission intensity of each cell asit passes through the instrument and generates a histogram plot of cellnumber versus fluorescent intensity. Gates or limits can be placed onthe histogram thus identifying a particular population of cells. In oneembodiment, the cell sorter is set up to select cells having the highestprobe intensity, usually a small fraction of the cells in the culture,and to separate these selected cells away from all the other cells. Thelevel of intensity at which the sorter is set and the fraction of cellswhich is selected, depend on the condition of the parent culture and thecriteria of the isolation. In general, the operator should first sort analiquot of the culture, and record the histogram of intensity versusnumber of cells. The operator can then set the selection level andisolate an appropriate number of the most active cells. Currently,fluorescence activated cell sorters are equipped with automated cellcloning devices. Such a device enables one to instruct the instrument tosingly deposit a selected cell into an individual growth well, where itis allowed to grow into a monoclonal culture. Thus, genetic homogeneityis established within the newly cloned culture.

IV. General Applications for the GFP Mutants

It should be self-evident that the mutant GFP and BFP sequencesdescribed here have unlimited uses, particularly as signal or reportersequences for the co-expression of other nucleic acid sequences ofinterest and/or to track the location and/or movement of other sequenceswithin the cell, within tissue and the like. For example, these reportertype sequences could be used to track the spread (or lack thereof) of adisease causal agent in drug screening assays or could readily be usedin diagnostics. Some of the more interesting applications are describedbelow.

A. Protein Trafficking

Normally, expressed mutant GFPs and BFPs are distributed throughout thecell (particularly mammalian cells), except for the nucleolus. However,as described below, when a GFP mutant is fused to the HIV-1 Rev protein,a hybrid molecule results which retains the Rev function and islocalized mainly in the nucleolus where Rev is found. Fusion to theN-terminal domain of the HIV-1 Nef protein produces a hybrid proteindetectable in the plasma membrane. Thus, the GFP mutants can be used tomonitor the subcellular targeting and transport of proteins to whichthey are fused.

B. Gene Therapy

The mutant GFPs described here have interesting and useful applicationsin gene therapy. Gene therapy in general is the correction of geneticdefects by insertion of exogenous cellular genes that encode a desiredfunction into cells that lack that function, such that the expression ofthe exogenous gene a) corrects a genetic defect or b) causes thedestruction of cells that are genetically defective. Methods of genetherapy are well known in the art, see, for example, Lu, M., et al.(1994), Human Gene Therapy 5:203; Smith, C. (1992), J. Hematotherapy1:155; Cassel, A., et al. (1993), Exp. Hematol. 21-:585 (1993); Larrick,J. W. and Burck, K. L., GENE THERAPY: APPLICATION OF MOLECULAR BIOLOGY,Elsevier Science Publishing Co., Inc., New York, N.Y. (1991) andKreigler, M. GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL, W. H.Freeman and Company, New York (1990), each incorporated herein byreference. One modality of gene therapy involves (a) obtaining from apatient a viable sample of primary cells of a particular cell type; (b)inserting into these primary cells a nucleic acid segment encoding adesired gene product; (c) identifying and isolating cells and cell linesthat express the gene product; (d) re-introducing cells that express thegene product; (e) removing from the patient an aliquot of tissueincluding cells resulting from step c and their progeny; and (f)determining the quantity of the cells resulting from step c and theirprogeny, in said aliquot. The introduction into cells in step c of apolycistronic vector that encodes GFP or BFP in addition to the desiredgene allows for the quick identification of viable cells that containand express the desired gene.

Another gene therapy modality involves inserting the desired nucleicacid into selected tissue cells in situ, for example into cancerous ordiseased cells, by contacting the target cells in situ with retroviralvectors that encode the gene product in question. Here, it is importantto quickly and reliably assess which and what proportion of cells havebeen transfected. Co-expression of GFP and BFP permits a quickassessment of proportion of cells that are transfected, and levels ofexpression.

C. Diagnostics

One potential application of the GFP/BFP variants is in diagnostictesting. The GFP/BFP gene, when placed under the control of promotersinduced by various agents, can serve as an indicator for these agents.Established cell lines or cells and tissues from transgenic animalscarrying GFP/BFP expressed under the desired promoter will becomefluorescent in the presence of the inducing agent.

Viral promoters which are transactivated by the corresponding virus,promoters of heat shock genes which are induced by various cellularstresses as well as promoters which are sensitive to organismalresponses, e.g. inflammation, can be used in combination with thedescribed GFP/BFP mutants in diagnostics.

In addition, the effect of selected culture conditions and components(salt concentrations, pH, temperature, trans-acting regulatorysubstances, hormones, cell-cell contacts, ligands of cell surface andinternal receptors) can be assessed by incubating cells in whichsequences encoding the fluorescent proteins provided herein are operablylinked to nucleic acids (especially regulatory elements such aspromoters) derived from a selected gene, and detecting the expressionand location of fluoresence.

D. Toxicology

Another application of the GFP/BFP-based methodologies is in the area oftoxicology. Assessment of the mutagenic potential of any compound is aprerequisite for its use. Until recently, the Ames assay in Salmonellaand tests based on chromosomal aberrations or sister chromatid exchangesin cultured mammalian cells were the main tools in toxicology. However,both assays are of limited sensitivity and specificity and do not allowstudies on mutation induction in various organs or tissues of the intactorganism.

The introduction of transgenic mice with a mutational target in ashuttle vector has made possible the detection of induced mutations indifferent tissues in vivo. The assay involves DNA isolation from tissuesof exposed mice, packaging of the target DNA into bacteriophage lambdaparticles and subsequent infection of E. coli. The mutational target inthis assay is either the lacZ or lacI genes and quantitation of blue vswhite plaques on the bacterial lawn allows for mutagenic assessment.

GFP/BFP could significantly simplify both the tissue culture andtransgenic mouse procedures. Expression of GFP/BFP under the control ofa repressor, which in turn is driven by the promoter of a constitutivelyexpressed gene, will establish a rapid method for evaluating themutagenic potential of an agent. The presence of fluorescent cells,following exposure of a cell line, tissue or whole animal carrying theGFP/BFP-based detection construct, will reflect the mutagenicity of thecompound in question. GFP/BFP expressed under the control of the targetDNA, the repressor gene, will only be synthesized when the repressor isinactivated or turned off or the repressor recognition sequences aremutated. Direct visualization of the detector cell line or tissue biopsycan qualitatively assess the mutagenicity of the agent, while FACS ofthe dissociated cells can provide for quantitative analysis.

E. Drug Screening

The GFP/BFP detection system could also significantly expedite andreduce the cost of some current drug screening procedures. A dual colorscreening system (DCSS), in which GFP is placed under the promoter of atarget gene and BFP is expressed from a constitutive promoter, couldprovide for rapid analysis of agents that specifically affect the targetgene. Established cell lines with the DCSS could be screened withhundreds of compounds in few hours. The desired drug will only influencethe expression of GFP. Non-specific or cytotoxic effects will bedetected by the second marker, BFP. The advantages of this system arethat no exogenous substances are required for GFP and BFP detection, theassay can be used with single cells, cell populations, or cell extracts,and that the same detection technology and instrumentation is used forvery rapid and non-destructive detection.

The search for antiviral agents which specifically block viraltranscription without affecting cellular transcription, could besignificantly improved by the DCSS. In the case of HIV, appropriate celllines expressing GFP under the HIV LTR and BFP under a cellularconstitutive promoter, could identify compounds which selectivelyinhibit HIV transcription. Reduction of only the green but not the bluefluorescent signal will indicate drug specificity for the HIV promoter.Similar approaches could also be designed for other viruses.

Furthermore, the search for antiparasitic agents could also be helped bythe DCSS. Established cell lines or transgenic nematodes or evenparasitic extracts where expression of GFP depends on parasite-specifictrans splicing sequences while BFP is under the control of host-specificcis splicing elements, could provide for rapid screen of selectiveantiparasitic drugs.

The invention will be more readily understood by reference to thefollowing specific examples which are included for purposes ofillustration only and are not intended to limit the invention unless sostated.

EXAMPLES

The following general protocol was used to generate mutant GFP- orBFP-encoding nucleic acids, transform host cells, and express the mutantGFP and BFP proteins:

Clone a nucleic acid that encodes either mwtGFP or BFP(Tyr₆₇ →His),under the control of eukaryotic or prokaryotic promoters, into astandard ds-DNA plasmid

Convert the plasmid vector to a ss-DNA by standard methods

Anneal the ss-DNA to 40-50 nucleotide DNA oligomers having basemismatches at the site(s) intended to be engineered

Convert the ss-DNA to a closed ds-DNA plasmid vector by use of DNApolymerase and standard protocols

Identify plasmids containing the desired mutations by restrictionanalysis following plasmid DNA isolation from E. coli strainstransformed with the mutagenized DNA

verify the presence of mutations by DNA sequencing

transfect human transformed embryonic kidney 293 cells with equalamounts of DNA from the appropriate plasmids

compare the fluorescence intensity of the signals

Nucleic acids and vectors

The mwtGFP cDNA (SEQ ID NO:1) was obtained from Dr. Chalfie of ColumbiaUniversity. All mutants described were obtained by modifying this mwtGFPsequence as detailed below.

The vectors used to clone and to express the GFPs and BFPs arederivatives of the commercially available plasmids pcDNA3 (Invitrogen,San Diego, Calif.), pBSSK+ (Stratagene, La Jolla, Calif.) and pET11a(Novagen, Madison, Wis.).

mwtGFP protein expression in mammalian cells

Several vectors for the expression of GFP in mammalian cells wereconstructed:

pFRED4 carries the mwtGFP sequences under the control of thecytomegalovirus (CMV) early promoter and the polyadenylation signal ofthe Human immunodeficiency Virus-1 (HIV) 3' Long Terminal Repeat (LTR).To derive pFRED4 we amplified the GFP coding sequence from plasmid #TU58(Chalfie et al., 1994) by the polymerase chain reaction (PCR). For PCRamplification of the GFP coding region, oligonucleotides #16417 and#16418 were used as primers. Oligonucleotide #16417:

    5'-GGAGGCGCGCAAGAAATGGCTAGCAAAGGAGAAGA-3'                  (SEQ ID NO:3),

containing the BssHII recognition sequence and the translationinitiation sequence of the HIV-1 Tat protein, was the sense primer. Theantisense primer, #16418:

    5'-GCGGGATCCTTATTTGTATAGTTCATCCATGCCATG-3'                 (SEQ ID NO:4)

contained the BamHI recognition sequence. The amplified fragment wasdigested with BssHII and BamHI and cloned into BssHII and BamHI digestedpCMV37M1-10D, a plasmid containing the CMV early promoter and the HIV-1p37gag region, followed by several cloning sites and the HIV-1 3' LTR.Thus the p37gag gene was replaced by GFP, resulting in pFRED4.

In a second step, the 1485 bp fragment from pFRED4, generated from StuIand BamHI double digestion, was subcloned into the 4747 bp vectorderived from the NruI and BamHI double digestion of pcDNA3. Theresulting plasmid, pFRED7 (SEQ ID NO:5), expresses GFP under the controlof the early CMV promoter and the bovine growth hormone polyadenylationsignal.

Bacterial expression

For bacterial expression, we constructed plasmid pBSGFP (SEQ ID NO:6), apBSSK+ derivative carrying mwtGFP. pBSGFP was generated by inserting theGFP containing region of pFRED4, digested with BamHII and BamHI andsubsequently treated with Klenow, into the EcoRV digested pBSSK+ vector.In pBSGFP the mwtGFP is fused downstream to the 43 amino acids of thealpha peptide of beta galactosidase, present in the pBSSK+ polylinkerregion. The added amino acids at the N-terminus of mwtGFP have noapparent effect on the GFP signal, as judged from subsequent plasmidscontaining precise deletions of the extra amino acids.

For GFP overexpression and purification we generated plasmid pFRED13(SEQ ID NO:7) by ligating the 717 bp fragment from pFRED7 digested withNheI and BamHI, to the 5644 bp fragment resulting from the NheI andBamHI double digestion of pET11a. In pFRED13, GFP is synthesized underthe control of the bacteriophage T7 phi10 promoter.

The oligonucleotides used for GFP mutagenesis were synthesized by theDNA Support Services of the ABL Basic Research Program of the NationalCancer Institute. DNA sequencing was performed by the PCR-assistedfluorescent terminator method (ReadyReaction DyeDeoxy Terminator CycleSequencing Kit, ABI, Columbia, Md.) according to the manufacturer'sinstructions. Sequencing reactions were resolved on the ABI Model 373ADNA Sequencing System. Sequencing data were analyzed using theSequencher program (Gene Codes, Ann Arbor, Mich.).

Enzymes were purchased from New England Biolabs (Beverly, Mass.) andused according to conditions described by the supplier. Chemicals usedfor the purification of wild type and mutant proteins were purchasedfrom SIGMA (St. Louis, Mo.). Tissue culture media were obtained fromBiofluids (Rockville, Md.) and GIBCO/BRL (Gaithersburg, Md.). Competentbacterial cells were purchased from GIBCO/BRL.

Preparation of mutants

Initially, plasmid pBSGFP was used to mutagenize the GFP coding sequenceby single-stranded DNA site directed mutagenesis, as described bySchwartz et al. (1992) J. Virol. 66:7176. In addition to changingspecific codons, our strategy was also to improve GFP expression byreplacing potential inhibitory nucleotide sequences without altering theGFP amino acid sequence. This approach has been successfully employed inthe past for other proteins (Schwartz et al. (1992) J. Virol. 66:7176)

For the pBSGFP mutagenesis the following oligonucleotides were used:

#17422 (SEQ ID NO:8):

    5'-CAATTTGTGTCCCAGAATGTTGCCATCTTCCTTGAAGTCAATACCTTT-3'

#17423 (SEQ ID NO:9):

    5'-GTCTTGTAGTTGCCGTCATCTTTGAAGAAGATGCTCCTTTCCTGTAC-3'

#17424 (SEQ ID NO:10):

    5'-CATGGAACAGGCAGTTTGCCAGTAGTGCAGATGAACTTCAGGGTAAGTTTTC-3'

#17425 (SEQ ID NO:11):

    5'-CTCCACTGACAGAGAACTTGTGGCCGTTAACATCACCATC-3'

#17426 (SEQ ID NO:12):

    5'-CCATCTTCAATGTTGTGGCGGGTCTTGAAGTTCACTTTGATTCCATT-3'

#17465 (SEQ ID NO:13):

    5'-CGATAAGCTTGAGGATCCTCAGTTGTACAGTTCATCCATGC-3'

Oligonucleotide #17426 introduces a mutation in GFP, converting theIsoleucine (lle) at position 168 into Threonine (Thr). The llel68Thrchange has been shown to alter the GFP spectrum and to also increase theintensity of GFP fluorescence by almost two-fold at the emission maxima(Heim et al. (1994), supra).

The mutagenesis mixture was used to transform DH5a competent E. colicells. Ampicilin resistant colonies were obtained and examined for theirfluorescent properties by excitation with UV light. One colony,significantly brighter than the rest, was apparent on the agar plate.This colony was further purified, the plasmid DNA was isolated and usedto transform DH5a competent bacteria. This time all the colonies werebright green when excited with the UV light, indicating that the brightgreen fluorescence was associated with the presence of the plasmid. Thesequence of the GFP segment (SEQ ID NO:14, representing only the segmentand not the whole plasmid) of this plasmid, called pBSGFPsg11, was thendetermined. The sequence analysis revealed that in addition to thedesigned nucleotide changes, which do no alter the amino acid sequenceof GFP, and the Ile168Thr mutation, a second spontaneous mutation hadoccurred. A thymidine at position 322 of SEQ ID NO:14, which is theGFP-coding region of the pPBSGFPsg11 DNA, was replaced by a cytosine.This nucleotide change converts the phenylalanine (Phe) at position 65of the GFP amino acid sequence into a leucine (Leu). A series ofexperiments, which will be described below, demonstrated that indeed thePhe65Leu mutation was responsible for the increase in the intensity ofthe fluorescent GFP signal.

In subsequent experiments, involving generation of rationally designedGFP mutant combinations to be detailed below, we also used thesingle-stranded DNA site directed mutagenesis approach. This time,however, the template DNAs were pFRED7 derivatives instead of pBSGFP.

Transfection and expression

The 293 cell line, an adenovirus-transformed human embryonal kidney cellline (Graham et al. (1977), J. Gen. Virol. 5:59) was used for proteinexpression analysis. The cells were cultured in Dulbecco's modifiedculture medium (DMEM) supplemented with 10% heat-inactivated fetalbovine serum (FBS, Biofluids).

Transfection was performed by the calcium phosphate coprecipitationtechnique as previously described (Graham et al. (1973), Virol. 52:456;Felber et al. (1990), J. Virol. 64:3734. Plasmid DNA was purified byQiagen columns according to the manufacturer's instructions (Qiagen). Amix of 5 to 10 μg of total DNA per ml of final precipitate was overlaidon the cells in 60 mm or 6- and 12-well tissue culture plates (Falcon),using 0.5, 0.25 and 0.125 ml of precipitate, respectively. Afterovernight incubation, the cells were washed, placed in medium withoutphenol red and measured in a plate spectrofluorometer, e.g., CytofluorII (Perceptive Biosystems, Framingham, Mass.)

Purification of wild-type and mutant proteins

E. coli strains carrying pFRED13 or other pET11a derivatives with mutantGFP genes were used for the overproduction and purification of the wtand mutant GFPs or BFPS. The cells were grown in 1 liter LB brothcontaining 100 μg/ml ampicillin at 32° C. to a density of 0.6-0.8optical density units at 600 nm. At this point, the cells were inducedwith 0.6 mM IPTG and incubated for four more hours. Following harvestingof the cell pellets, cellular extracts were prepared as described byJohnson, B. H and Hecht, M. H., 1994, Biotechnol. 12: 1357.

GFPs and BFPs were purified from the cellular extracts as follows:Ammonium sulfate (AS) was added first to the extracts (50 g AS per 100 gsupernatant) to precipitate the proteins. The precipitants werecollected by centrifugation at 7500×g for 15 min and the pellets weredissolved in 5ml of 1 M AS. The samples were then loaded onphenylsepharose column (HR10/10, Pharmacia, Piscataway, N.J.) and washedwith 20 mM 2-[N-morpholino]ethanesulfonic Acid (MES) pH 5.6 and 1 M AS.Proteins were eluted with a 45 ml gradient to 20 mM MES, pH 5.6.Fractions containing the GFP or BFP protein were colored even undervisible light.

Green or blue-colored fractions were further purified on Q-sepharose(Mono Q, HR5/5, Pharmacia) with a 20 ml gradient from 20 mM Tris pH 7.0to 20 mM Tris pH 7.0, 0.25 M NaCl.

The AS precipitation step was performed at 4° C. while thechromatographic procedures were performed at room temperature.

Determination of protein concentration

Protein concentrations were determined using the commercially availableBradford protein assay (BioRad, Hercules, Calif.) with bovine IgGprotein as a standard.

Analytical polyacrylamide gels

Analytical polyacrylamide gel electrophoresis was used to visualize thedegree of purity of the purified GFP or BFP proteins. In all cases, 1 mmthick, 12' acrylamide gels (containing 0.1% SDS, in Tris buffer, pH 7.4)were used, and electrophoresis was performed for 2 hours at 120 V. Gelswere stained with Coomassie Blue to visualize the proteins.

Fluorescence measurements

Excitation and emission spectra of solutions of the fluorescent proteinswere obtained using a Perkin Elmer L550B spectrofluorimeter (PerkinElmer, Advanced Biosystems, Foster City,, Calif.).

The relative fluorescence data for the GFP mutants in Table I below wereobtained by comparing the cellular fluorescence of the GFP mutantsexpressed in the transformed human embryonic kidney cell line 293 withmwtGFP expressed in the same cell line. Likewise, the relativefluorescence data for the BFP mutants in Table I below were obtained bycomparing the cellular fluorescence of the BFP mutants expressed in 293cells with BFP(Tyr₆₇ →His) expressed in the same cell line. Equalamounts of DNA encoding modified wild type or mutant proteins wereintroduced into 293 cells. Cellular fluorescence was quantified 24 h or48 hr. post-transfection using Cytofluor II.

A list of GFP mutant proteins indicating the introduced amino acidmutations is shown in Table I.

                  TABLE I                                                         ______________________________________                                        GFP and BFP mutants                                                                     Amino Acid Position                                                 PROTEIN   65    66       67  164    168  239                                  ______________________________________                                        mwt GFP   F     S        Y   V      I    K                                      SG12 L                                                                        SG11 L    T N                                                                 SG25 L C   T N                                                                BFP   H                                                                       SB42 L  H                                                                     SB49   H A                                                                    SB50 L  H A                                                                 ______________________________________                                    

EXAMPLE 1

SG12

A number of the unique mutants described herein derive from thediscovery of an unplanned and unexpected mutation called "SG12",obtained in the course of site-directed mutagenesis experiments, whereina phenylalanine at position 65 of mwtGFP was converted to leucine. SG12was prepared as follows: Two plasmids carrying SG12 (SEQ ID NO:15) weregenerated, pFRED12 for expression in mammalian cells, and pFRED16 forexpression in E. coli and protein purification. pFRED12 was constructedby ligating the 1557 bp fragment from the double digestion of pFRED7with Avr II and Pml I into the 4681 bp fragment generated from the AvrII and Pml I digestion of pFRED11 (see below). pFRED16 was derived bysubcloning the 717 bp segment resulting from the digestion of pFRED12with NheI and BamHI to the 5644 bp fragment of the pET11a vectordigested with the same restriction enzymes.

The specific activity of SG12 was about 9-12 times that of mwtGFP. SeeTable II.

EXAMPLE 2

SG11

A mutant referred to as "SG11," which combined the phenylalanine 65 toleucine alteration with an isoleucine 168 to threonine substitution anda lysine 239 to asparagine susbstitution, gave a further enhancedfluorescence intensity. SG11 was prepared as follows: Two plasmidscarrying SG11 (SEQ ID NO:16) were generated: pFRED11 for expression inmammalian cells and pFRED15 for expression in E. coli and proteinpurification. pFRED11 was constructed by ligating the 717 bp region frompBSGFPsg11 DNA digested with NheI and BamHI to the 5221 bp fragmentderived from the digestion of pFRED7 with the same enzymes. pFRED15 wasgenerated by subcloning the 717 bp segment resulting from the digestionof pFRED11 with NheI and BamHI to the 5644 bp fragment of the pET11avector, digested with the same restriction enzymes.

The mutant SG11 encodes an engineered GFP wherein the alterationcomprises the conversion of phenylalanine 65 to leucine and theconversion of isoleucine 168 to threonine. The additional alteration ofthe C-terminal lys 239 to asn is without effect; the C-terminal lys orasn may be deleted without affecting fluorescence. The specific activityof SG11 is about 19-38 times that of mwtGFP. See Table II.

EXAMPLE 3

SG25

A third and further improved GFP mutant was obtained by further mutating"SG11." This mutant is referred to as "SG25" and comprises, in addtionto the SG11 substitutions, and additional substitution of a cysteine forthe serine normally found at position 66 in the sequence. SG11 wasprepared as follows: Two plasmids carrying SG25 (SEQ ID NO:17) weregenerated: pFRED25 for expression in mammalian cells and pFRED63 forexpression in E. coli and protein purification. pFRED25 was constructedby site directed mutagenesis of pFRED11, using oligonucleotide #18217(SEQ ID NO:18): 5'-CATTGAACACCATAGCACAGAGTAGTGACTAGTGTTGGCC-3'. Thisoligonucleotide incorporates the Ser66Cys mutation into SG11. Ser66Cyshad been shown to both alter the GFP excitation maxima withoutsignificant change in the emission spectrum and to also increase theintensity of the fluorescent signal of GFP (Heim et al., 1995).

pFRED63 was generated by subcloning the 717 bp segment resulting fromthe digestion of pFRED25 with NheI and BamHI to the 5644 bp fragment ofthe pET11a vector, digested with the same restriction enzymes.

The mutant SG25 encodes an engineered GFP wherein the alterationcomprises the conversion of phenylalanine 65 to leu, the conversion ofisoleucine 168 to threonine and the conversion of serine 66 to cysteine.As with SG11, the additional alteration of the C-terminal lysine 239 toasparagine is without effect; the C-terminal lysine or aspragine may bedeleted without affecting fluorescence. The specific activity of SG25 isabout 56 times that of mwtGFP. See Table II.

EXAMPLE 4

Additional green fluorescent mutants

Additional alterations at different amino acids of the mwtGFP, whencombined with SG11 and SG25, yielded proteins having at least 5× greatercellular fluorescence compared to the mwtGFP. A non-limiting list ofthese mutations is provided below:

    ______________________________________                                        GFP variants with enhanced cellular fluorescence                                    Protein    Altered Amino Acids                                          ______________________________________                                        SG20         F65L, S66T, I168T, K239N                                           SG21 F65L, S66A, I168T, K239N                                                 SG27 Y40L, F65L, I168T, K239N                                                 SG30 F47L, F65L, I168T, K239N                                                 SG32 F72L, F65L, I168T, K239N                                                 SG43 F65L, I168T, Y201L, K239N                                                SG46 F65L, V164A, I168T, K239N                                                SG72 F65L, S66C, V164A, I168T, K239N                                          SG91 F65L, S66C, F100L, I168T, K239N                                          SG94 F65L, S66C, Y107L, I168T, K239N                                          SG95 F65L, S66C, F115L, I168T, K239N                                          SG96 F65L, S66C, F131L, I168T, K239N                                          SG98 F65L, S66C, Y146L, I168T, K239N                                          SG100 F65L, S66C, Y152L, I168T, K239N                                         SG101 F65L, S66C, I168T, Y183L, K239N                                         SG102 F65L, S66C, I168T, F224L, K239N                                         SG103 F65L, S66C, I168T, Y238L, K239N                                         SG106 F65L, S66T, V164A, I168T, K239N                                       ______________________________________                                    

EXAMPLE 5

SB42

The blue fluorescent proteins described here and below were derived fromthe known GFP mutant (Heim et al., PNAS, 1994) wherein histidine issubstituted for tyrosine at position 67. We have designated this knownmutant BFP(Tyr₆₇ →His). BFP(Tyr₆₇ →His) has a shifted emission spectrum.It emits blue light, i.e., it is a blue fluorescent protein (BFP).

By introducing the same mutation in BFP(Tyr₆₇ →His) that was used togenerate SG12, i.e., leucine for phenylalanine at position 65, wecreated a new mutant that has unexpectedly high fluorescence that werefer to as "SuperBlue-42" (SB42). SB42 was prepared as follows: Twoplasmids carrying SB42 (SEQ ID NO:19) were generated: pFRED42 forexpression in mammalian cells and pFRED65 for expression in E. coli andprotein purification. pFRED42 was constructed by site directedmutagenesis of pFRED12, using oligonucleotide #bio25(5-CATTGAACACCATGAGAGAGAGTAGTGACTAGTGTTGGCC-3') (SEQ ID NO:20). Thisoligonucleotide incorporates the Tyr₆₇ →His mutation into SG12, thusgenerating the Phe65Leu, Tyr₆₇ →His double mutant.

pFRED65 was created by subcloning the 717 bp segment resulting from thedigestion of pFRED42 with NheI and BamHI to the 5644 bp fragment of thepET11a vector, digested with the same restriction enzymes.

The mutant SB42 encodes an engineered BFP wherein the alterationscomprise the conversion of tyrosine 67 to histidine and the conversionof phenylalanine 65 to leucine. The specific activity of SB42 is about27 times that of BFP(Tyr₆₇ →His). See Table II.

EXAMPLE 6

SB49

An independent mutation of BFP(Tyr₆₇ →His) which substitutes the valineat position 164 with an alanine is referred to as "SB49." SB49 wasprepared as follows: Plasmid pFRED49 expresses SB49 (SEQ ID NO:21) inmammalian cells. pFRED49 was generated by site directed mutagenesis ofpFRED12, using oligonucleotides #19059 and #bio24. Oligonucleotide#19059 (5'-CTTCAATGTTGTGGCGGATCTTGAAGTTCGCTTTGATTCCATTC-3') (SEQ IDNO:22) introduces the Val164Ala mutation in SG12 while oligonucleotide#bio24 (5'-CATTGAACACCATGAGAGAAAGTAGTGACTAGTGTTGGCC-3') (SEQ ID NO:23)reverts the Phe65Leu alteration to the wt sequence and, at the sametime, incorporates the Tyr₆₇ →His mutation.

The mutant SB49 encodes an engineered BFP wherein the alterationscomprise the conversion of tyrosine 67 to histidine, and the conversionof valine 164 to alanine. The specific activity of SB49 was about 37times that of BFP(Tyr₆₇ →His). See Table II.

EXAMPLE 7

SB50

A combination of the above two BFP mutations resulted in "SB50," whichgave an even greater fluorescence enhancement than either of theprevious mutations. SB50 was prepared as follows: Two plasmids carryingSB50 (SEQ ID NO: 24) were generated: pFRED50 for expression in mammaliancells and pFRED67 for expression in E. coli and protein purification.pFRED50 was constructed by site directed mutagenesis of pFRED12, usingoligonucleotides #19059 and #bio25.

pFRED67 was created by subcloning the 717 bp segment resulting from thedigestion of pFRED50 with NheI and BamHI to the 5644 bp fragment of thepET11a vector digested with the same restriction enzymes.

The mutant SB50 encodes an engineered BFP wherein the alterationscomprise the conversion of tyrosine 7 to histidine, the conversion ofphenylalanine 65 to leucine and the conversion of alanine 164 to valine.The specific activity of SB50 was about 63 times that of BFP(Tyr₆₇ →His)See Table II.

                  TABLE II                                                        ______________________________________                                                                   Factor of                                               increased Factor of                                                           green increased blue                                                          fluorescence fluorescence                                                     (at maximum (at maximum                                                     Excitation Emission emission) as emission) as                                 Maximum Maximum compared to compared to                                      Mutant (nm) (nm) mwt.GFP BFP (Tyr                                                                              .sub.67 →His)                       ______________________________________                                        SG12   398       509        9-12X                                               SG11 471 508 19-38X                                                           SG25 473 509 50-100X                                                          SB42 387 450  27X                                                             SB49 387 450  37X                                                             SB50 387 450  63X                                                           ______________________________________                                    

The dramatic increase in fluorescent activity resulting from the aminoacid substitutions of the present invention was wholly unexpected. Thecellular fluorescence of the mutants was at least five times greater,and usually over twenty times greater, than that of the parent mwtGFP orBFP(Tyr₆₇ →His). Note that the maximum emission wavelengths vary amongthe mutants, and that the above-reported fold increases refer only tominimal increases in relative cellular fluorescence at the maximumemission wavelength of the mutant. Given a particular wavelength, thevalues may be substantially larger, i.e., the mutants may have a200-fold greater cellular fluorescence than the reference mwtGFP orBFP(Tyr₆₇ →His). This is important because devices for measuringfluorescence often have set wavelengths, or the limitations of a givenexperiment often require the use of a set wavelength. Thus, for example,the emission and detection parameters of a fluorescence microscope or afluorescence-activated cell sorter may be set for a wavelength whereinthe cellular fluorescence of a given mutant is 200-fold greater thanthat of the known GFPs and BFPS.

The GFP and BFP mutants of this invention, in contrast to the wild typeprotein or other reported mutants, allow detection of green fluorescencein living mammalian cells when present in few copies stably integratedinto the genome. This high cellular fluorescence of the mutant GFPs andBFPs is useful for rapid and simple detection of gene expression inliving cells and tissues and for repeated analysis of gene expressionover time under a variety of conditions. They are also useful for theconstruction of stable marked cell lines that can be quickly identifiedby fluorescence microscopy or fluorescence activated cell sorting.

EXAMPLE 8

We have established fluoroplate-based assays for the quantitation ofgene expression after transfections. In a number of embodiments, anucleic acid encoding a mutant GFP or BFP of this invention is insertedinto a vector and introduced into and expressed in a cell. Typically,expression of GFP mutants can be detected as quickly as 5 hourspost-infection or less. Expression is followed over time in living cellsby a simple measurement in multi-well plates. In this way, manytransfections can be processed in parallel.

EXAMPLE 9

The vectors and nucleic acids provided herein are used to generatechimeric proteins wherein a nucleic acid sequence that encodes aselected gene product is fused to the C- or N-terminus of the mutantGFPs and/or BFPs of this invention. A number of unique viral, plasmidand hybrid gene constructs have been generated that incorporate the newmutant GFP and/or mutant BFP sequences indicated above. These include:

HIV viral sequences (in the nef gene) containing SG11 or SG25

Neomycin & hygromycin plasmids containing SG11 or SG25

Moloney Leukemia Virus vector (retrovirus) also expressing SG25

Hybrid gene constructs expressing HIV viral proteins (rev, td-rev, tat,nef, gag, env, and vpr) and either SG11 or SG25 or SB50.

Hybrid gene construct containing vectors that incorporate thecytoplasmic proteins ran, B23, nucleolin, poly-A binding protein andeither SG11 or SG25 or SB50.

These hybrids of the mutant nucleic acids provided herein are used tostudy protein trafficking in living mammalian cells. Like the wild typeGFP, the mutant GFP proteins are normally distributed throughout thecell except for the nucleolus. Fusions to other proteins redistributethe fluorescence, depending on the partner in the hybrid. For example,fusion with the entire HIV-1 Rev protein results in a hybrid moleculewhich retains the Rev function and is localized in the nucleolus whereRev is preferentially found. Fusion to the N-terminal domain of theHIV-1 Nef protein created a chimeric protein detected in the plasmamembrane, the site of Nef localization.

EXAMPLE 10

pCMVgfo11

pCMVgfo11 is a pFRED11 derivative containing the bacterial neomycinphosphotransferase gene (neo) (Southern and Berg (1982) J. Mol. Appl.Genetics 1:327) fused at the C-terminus of SG11. A four amino acid(Gly-Ala-Gly-Ala) (SEQ ID NO:26) linker region connects the last aminoacid of SG11 to the second amino acid of neo, thus generating the hybridSG11-neo protein (gfo11, SEQ ID NO:25). Gfo11 is expressed from the CMVpromoter and contains the intact SG11 polypeptide and all of neo exceptfor the first Met. pCMVgfo11 was constructed in several steps. First,pFRED11DNae was constructed by NaeI digestion of pFRED11 andself-ligation of the 4613 bp fragment. The NaeI deletion removes theSV40 promoter and neo gene from pFRED11, thus creating pFRED11DNae.Next, in order to fuse the neo coding region downstream to SG11, the neogene was PCR amplified from pcDNA3 using primers Bio51(5'-CGCGGATCCTTCGAACAAGATGGATTGCACGC-3') (SEQ ID NO:27) and Bio52(5-CCGGAATTCTCAGAAGAACTCGTCAAGAAGGCGA-3') (SEQ ID NO:28). Primer Bio51introduces a BamHI site followed by a BstBI recognition sequence at the5' end of neo, while primer Bio52 introduces an EcoRI site 3' to the neogene. The PCR product was digested with BamHI and EcoRI and cloned intothe 4582 bp vector resulting from the BamHI-EcoRI digestion ofpFRED11DNae, thus generating pFRED11DNaeBstNeo. Subsequently, SG11 wasPCR amplified from pFRED11DNae using primers Bio49(5'-GGCGCGCAAGAAATGGCTAGCAAAGGAGAAGAACTCTTCACTGGAG-3') (SEQ ID NO:29)and Bio50 (5'-CCCATCGATAGCACCAGCACCGTTGTACAGTTCATCCATGCCATGT-3') (SEQ IDNO:30) to remove the sgII stop codon in pFRED11DNaeBstNeo and tointroduce the four amino acid (Gly-Ala-Gly-Ala) linker followed by aClaI site. The PCR product was digested with NheI and ClaI and clonedinto the 4763 bp NhelBstBi fragment from pFRED11DNaeBstNeo, thusgenerating pCMVgfo11.

Following transfection of 293 cells (Graham et al. (1977), J. Gen.Virol. 5:59) as well as other human and mouse cell lines with pCMVgfo11,bright fluorescent transfectants were apparent under the flourescentmicroscope and colonies resistant to G418 could be obtained two weekslater.

It should be noted that pCMVgfo11 was the best protein fusion in termsof fluorescent emission intensity and number of G418 resistant coloniescompared to several SG11-neo or neo-SG11 fusions generated and examined.

EXAMPLE 11

pPGKgfo25

pPGKgfo25 is a pCMVgfoII derivative containing SG25 instead of SG11within gfo (SEQ ID NO: 31). Expression of gfo25 in pPGKgfo25 is underthe control of the mouse phosphoglycerate kinase-1 (PGK) promoter.

pPGKgfo25 was constructed in several steps. First, a SacII site wasintroduced downstream of the PGK promoter in pPGKneobpA (Soriano et al.(1991) Cell: 64-393) by:

i) annealing oligonucleotides #18990 (SEQ ID NO:32)(5'-GACCGGGACACGTATCCAGCCTCCGC-3') and 18991 (SEQ ID NO:33)(5'-GGAGGCTGGATACGTGTCCCGGTCTGCA-3') to create a double stranded adapterfor PstI at the 5' end and SacII at the 3' end.

ii) ligating this adapter to the 3423 bp fragment from the PstI-SacIIdouble digestion of pPGKneobpA, thus generating pPGKPtAfSc.

Next, the CMV promoter of pFRED25 was replaced with the PGK promoter bycloning the 565 bp SaII (filled with Klenow)-SacII region frompPGKPtAfSc to the 5288 bp BgIII (filled with Klenow)-SacII fragment frompFRED25, resulting in pFRED25PGK. In the final step, pPGKgfo25 wasconstructed by ligating the 813 bp BgIII-NdeI fragment from pFRED25PGKcontaining the PGK promoter and SG25, to the 4185 bp BgIII-NdeI fragmentof pCMVgfo11.

EXAMPLE 12

pGen-PGKgfo25RO (SEQ ID NO: 34)

pGen-PGKgfo25RO is a pGen- (Soriano et al. (1991), J. Virol. 65:2314)derivative containing the gfo25 hybrid under the control of PGKpromoter. It was constructed by subcloning the 2810 bp SaII fragment ofpPGKgfo25 into the XhoI site of pGen. In viruses generated frompGen-PGKgfo25RO (see below) transcription originated from the PGKpromoter is in reverse orientation (RO) to that initiated from the virallong terminal repeats (LTR).

To generate ecotropic or pseudotyped viruses, pGen-PGKgfo25RO wasco-transfected into 293 cells together with pHIT60 and pHIT123 DNAs(production of ecotropic virus) or with pHIT60 and pHCMV-G DNAs(production of pseudotyped virus). pHIT60 and pHIT123 contain thegag-pol and env coding regions from the Moloney murine leukemia virus(Mo-MLV) respectively, under the control of the CMV promoter (Soneoka etal. (1995), Nuc. Acid Res. 23:628. pHCMV-G contains the coding region ofthe G protein from the vesicular stomatitis virus (VSV) expressed fromthe CMV promoter (Yee et al. (1994), Proc. Nat'l Acad. Sci. USA 91:9564.Virus-containing supernatants were harvested 48 hours post transfection,filtered and stored at -80° C.

EXAMPLE 13

pNLnSG11 (SEQ ID NO:35)

The SG11 sequence from plasmid pFRED11 was PCR-amplified with primers#17982 (SEQ ID NO:36)(5'-GGGGCGTACGGAGCGCTCCGAATTCGGTACCGTTTAAACGGGCCCTCTCGAGTCCGTTGTACAGTTCATCCATG-3') and #17983 (SEQ ID NO:37)(5'-GGGGGAATTCGCGCGCGTACGTAAGCGCTAGCTGAGCAAGAAATGGCTAGCAAAGGAGAAGAACTC-3'). The PCR product was digested with BlpI and XhoI andcloned into the large BlpI-XhoI fragment from pNL4-3 (Adachi et al.(1986), J. Virol. 59: 284. In pNLnSG11 the full SG11 polypeptidecontaining an additional four linker-encoded amino acids at theC-terminus, is expressed as a hybrid protein with the 24 N-terminalamino acids of the HIV-1 protein Nef.

We constructed transmissible HIV-1 stocks with our mutants, whichgenerate green fluorescence upon transfection of human cells. Thesetransmissible HIV-1 stocks are used to detect the kinetics of infectionunder a variety of conditions. In particular, they are used to study theeffects of drugs on the kinetics of infection. The level offluorescence, and the subcellular compartmentalization of thatfluorescence, is easily visualized and quantified using well knownmethods. This system is easy to visualize, and dramatically cuts thecosts of many experiments that are presently tedious and expensive.

To produce infectious virus, pNLnSG11 was transfected in 293 cells. 24hours later, Jurkat cells were added to the transfectants. At varioustimes post-infection, the medium was removed, filtered, and used toinfect fresh Jurkat or other HIV-1-permissive cells. Two days later theinfected cells were green under fluorescent microscope. Visible syncytiawere also green. Viral stocks were generated and kept at -80° C.

When the nucleic acids, vectors, mutant proteins provided herein arecombined with the knowledge of those skilled in the art of geneticengineering and the guidance provided herein, it will be apparent to oneof ordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein. These changes and modifications are encompassed bythe present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 37                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/product= "wild type Aequorea           victoria                                                                                       Green Flu - #orescent Protein (wtGF)"                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG GCT AGC AAA GGA GAA GAA CTC TTC ACT GG - #A GTT GTC CCA ATT        CTT       48                                                                    Met Ala Ser Lys Gly Glu Glu Leu Phe Thr Gl - #y Val Val Pro Ile Leu            1               5 - #                 10 - #                 15              - - GTT GAA TTA GAT GGT GAT GTT AAT GGG CAC AA - #A TTT TCT GTC AGT GGA           96                                                                       Val Glu Leu Asp Gly Asp Val Asn Gly His Ly - #s Phe Ser Val Ser Gly                        20     - #             25     - #             30                  - - GAG GGT GAA GGT GAT GCA ACA TAC GGA AAA CT - #T ACC CTT AAA TTT ATT          144                                                                       Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Le - #u Thr Leu Lys Phe Ile                    35         - #         40         - #         45                      - - TGC ACT ACT GGA AAA CTA CCT GTT CCA TGG CC - #A ACA CTT GTC ACT ACT          192                                                                       Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pr - #o Thr Leu Val Thr Thr                50             - #     55             - #     60                          - - TTC TCT TAT GGT GTT CAA TGC TTT TCA AGA TA - #C CCG GAT CAT ATG AAA          240                                                                       Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Ty - #r Pro Asp His Met Lys            65                 - # 70                 - # 75                 - # 80       - - CGG CAT GAC TTT TTC AAG AGT GCC ATG CCC GA - #A GGT TAT GTA CAG GAA          288                                                                       Arg His Asp Phe Phe Lys Ser Ala Met Pro Gl - #u Gly Tyr Val Gln Glu                            85 - #                 90 - #                 95              - - AGA ACT ATA TTT TTC AAA GAT GAC GGG AAC TA - #C AAG ACA CGT GCT GAA          336                                                                       Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Ty - #r Lys Thr Arg Ala Glu                       100      - #           105      - #           110                  - - GTC AAG TTT GAA GGT GAT ACC CTT GTT AAT AG - #A ATC GAG TTA AAA GGT          384                                                                       Val Lys Phe Glu Gly Asp Thr Leu Val Asn Ar - #g Ile Glu Leu Lys Gly                   115          - #       120          - #       125                      - - ATT GAT TTT AAA GAA GAT GGA AAC ATT CTT GG - #A CAC AAA TTG GAA TAC          432                                                                       Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gl - #y His Lys Leu Glu Tyr               130              - #   135              - #   140                          - - AAC TAT AAC TCA CAC AAT GTA TAC ATC ATG GC - #A GAC AAA CAA AAG AAT          480                                                                       Asn Tyr Asn Ser His Asn Val Tyr Ile Met Al - #a Asp Lys Gln Lys Asn           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GGA ATC AAA GTT AAC TTC AAA ATT AGA CAC AA - #C ATT GAA GAT GGA        AGC      528                                                                    Gly Ile Lys Val Asn Phe Lys Ile Arg His As - #n Ile Glu Asp Gly Ser                          165  - #               170  - #               175              - - GTT CAA CTA GCA GAC CAT TAT CAA CAA AAT AC - #T CCA ATT GGC GAT GGC          576                                                                       Val Gln Leu Ala Asp His Tyr Gln Gln Asn Th - #r Pro Ile Gly Asp Gly                       180      - #           185      - #           190                  - - CCT GTC CTT TTA CCA GAC AAC CAT TAC CTG TC - #C ACA CAA TCT GCC CTT          624                                                                       Pro Val Leu Leu Pro Asp Asn His Tyr Leu Se - #r Thr Gln Ser Ala Leu                   195          - #       200          - #       205                      - - TCG AAA GAT CCC AAC GAA AAG AGA GAC CAC AT - #G GTC CTT CTT GAG TTT          672                                                                       Ser Lys Asp Pro Asn Glu Lys Arg Asp His Me - #t Val Leu Leu Glu Phe               210              - #   215              - #   220                          - - GTA ACA GCT GCT GGG ATT ACA CAT GGC ATG GA - #T GAA CTA TAC AAA TAA          720                                                                       Val Thr Ala Ala Gly Ile Thr His Gly Met As - #p Glu Leu Tyr Lys  *            225                 2 - #30                 2 - #35                 2 -      #40                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 239 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ala Ser Lys Gly Glu Glu Leu Phe Thr Gl - #y Val Val Pro Ile        Leu                                                                               1               5 - #                 10 - #                 15             - - Val Glu Leu Asp Gly Asp Val Asn Gly His Ly - #s Phe Ser Val Ser Gly                   20     - #             25     - #             30                  - - Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Le - #u Thr Leu Lys Phe Ile               35         - #         40         - #         45                      - - Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pr - #o Thr Leu Val Thr Thr           50             - #     55             - #     60                          - - Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Ty - #r Pro Asp His Met Lys       65                 - # 70                 - # 75                 - # 80       - - Arg His Asp Phe Phe Lys Ser Ala Met Pro Gl - #u Gly Tyr Val Gln Glu                       85 - #                 90 - #                 95              - - Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Ty - #r Lys Thr Arg Ala Glu                  100      - #           105      - #           110                  - - Val Lys Phe Glu Gly Asp Thr Leu Val Asn Ar - #g Ile Glu Leu Lys Gly              115          - #       120          - #       125                      - - Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gl - #y His Lys Leu Glu Tyr          130              - #   135              - #   140                          - - Asn Tyr Asn Ser His Asn Val Tyr Ile Met Al - #a Asp Lys Gln Lys Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Ile Lys Val Asn Phe Lys Ile Arg His As - #n Ile Glu Asp Gly        Ser                                                                                             165  - #               170  - #               175             - - Val Gln Leu Ala Asp His Tyr Gln Gln Asn Th - #r Pro Ile Gly Asp Gly                  180      - #           185      - #           190                  - - Pro Val Leu Leu Pro Asp Asn His Tyr Leu Se - #r Thr Gln Ser Ala Leu              195          - #       200          - #       205                      - - Ser Lys Asp Pro Asn Glu Lys Arg Asp His Me - #t Val Leu Leu Glu Phe          210              - #   215              - #   220                          - - Val Thr Ala Ala Gly Ile Thr His Gly Met As - #p Glu Leu Tyr Lys          225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..35                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide sense primer                     #16417"                                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGAGGCGCGC AAGAAATGGC TAGCAAAGGA GAAGA       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..36                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide antisense                        primer #- #16418"                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GCGGGATCCT TATTTGTATA GTTCATCCAT GCCATG      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6238 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..6238                                                         (D) OTHER INFORMATION: - #/note= "pFRED7"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TG -             #CTCTGATG     60                                                                 - - CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GA -            #GTAGTGCG    120                                                                 - - CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AA -            #GAATCTGC    180                                                                 - - TTAGGGTTAG GCGTTTTGCG CTGCTTCGCC TCGAGGCCTG GCCATTGCAT AC -            #GTTGTATC    240                                                                 - - CATATCATAA TATGTACATT TATATTGGCT CATGTCCAAC ATTACCGCCA TG -            #TTGACATT    300                                                                 - - GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AG -            #CCCATATA    360                                                                 - - TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CC -            #CAACGACC    420                                                                 - - CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GG -            #GACTTTCC    480                                                                 - - ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CA -            #TCAAGTGT    540                                                                 - - ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GC -            #CTGGCATT    600                                                                 - - ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GT -            #ATTAGTCA    660                                                                 - - TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TA -            #GCGGTTTG    720                                                                 - - ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TT -            #TTGGCACC    780                                                                 - - AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CA -            #AATGGGCG    840                                                                 - - GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCG TTTAGTGAAC CG -            #TCAGATCG    900                                                                 - - CCTGGAGACG CCATCCACGC TGTTTTGACC TCCATAGAAG ACACCGGGAC CG -            #ATCCAGCC    960                                                                 - - TCCGCGGGCG CGCAAGAAAT GGCTAGCAAA GGAGAAGAAC TCTTCACTGG AG -            #TTGTCCCA   1020                                                                 - - ATTCTTGTTG AATTAGATGG TGATGTTAAT GGGCACAAAT TTTCTGTCAG TG -            #GAGAGGGT   1080                                                                 - - GAAGGTGATG CAACATACGG AAAACTTACC CTTAAATTTA TTTGCACTAC TG -            #GAAAACTA   1140                                                                 - - CCTGTTCCAT GGCCAACACT TGTCACTACT TTCTCTTATG GTGTTCAATG CT -            #TTTCAAGA   1200                                                                 - - TACCCGGATC ATATGAAACG GCATGACTTT TTCAAGAGTG CCATGCCCGA AG -            #GTTATGTA   1260                                                                 - - CAGGAAAGAA CTATATTTTT CAAAGATGAC GGGAACTACA AGACACGTGC TG -            #AAGTCAAG   1320                                                                 - - TTTGAAGGTG ATACCCTTGT TAATAGAATC GAGTTAAAAG GTATTGATTT TA -            #AAGAAGAT   1380                                                                 - - GGAAACATTC TTGGACACAA ATTGGAATAC AACTATAACT CACACAATGT AT -            #ACATCATG   1440                                                                 - - GCAGACAAAC AAAAGAATGG AATCAAAGTT AACTTCAAAA TTAGACACAA CA -            #TTGAAGAT   1500                                                                 - - GGAAGCGTTC AACTAGCAGA CCATTATCAA CAAAATACTC CAATTGGCGA TG -            #GCCCTGTC   1560                                                                 - - CTTTTACCAG ACAACCATTA CCTGTCCACA CAATCTGCCC TTTCGAAAGA TC -            #CCAACGAA   1620                                                                 - - AAGAGAGACC ACATGGTCCT TCTTGAGTTT GTAACAGCTG CTGGGATTAC AC -            #ATGGCATG   1680                                                                 - - GATGAACTAT ACAAATAAGG ATCCACTAGT AACGGCCGCC AGTGTGCTGG AA -            #TTCTGCAG   1740                                                                 - - ATATCCATCA CACTGGCGGC CGCTCGAGCA TGCATCTAGA GGGCCCTATT CT -            #ATAGTGTC   1800                                                                 - - ACCTAAATGC TAGAGCTCGC TGATCAGCCT CGACTGTGCC TTCTAGTTGC CA -            #GCCATCTG   1860                                                                 - - TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC AC -            #TGTCCTTT   1920                                                                 - - CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT AT -            #TCTGGGGG   1980                                                                 - - GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CA -            #TGCTGGGG   2040                                                                 - - ATGCGGTGGG CTCTATGGCT TCTGAGGCGG AAAGAACCAG CTGGGGCTCT AG -            #GGGGTATC   2100                                                                 - - CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG CG -            #CAGCGTGA   2160                                                                 - - CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TC -            #CTTTCTCG   2220                                                                 - - CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG CATCCCTTTA GG -            #GTTCCGAT   2280                                                                 - - TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA GGGTGATGGT TC -            #ACGTAGTG   2340                                                                 - - GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG TT -            #CTTTAATA   2400                                                                 - - GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT TC -            #TTTTGATT   2460                                                                 - - TATAAGGGAT TTTGGGGATT TCGGCCTATT GGTTAAAAAA TGAGCTGATT TA -            #ACAAAAAT   2520                                                                 - - TTAACGCGAA TTAATTCTGT GGAATGTGTG TCAGTTAGGG TGTGGAAAGT CC -            #CCAGGCTC   2580                                                                 - - CCCAGGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AG -            #GTGTGGAA   2640                                                                 - - AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TA -            #GTCAGCAA   2700                                                                 - - CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TC -            #CGCCCATT   2760                                                                 - - CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GC -            #CTCTGCCT   2820                                                                 - - CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TG -            #CAAAAAGC   2880                                                                 - - TCCCGGGAGC TTGTATATCC ATTTTCGGAT CTGATCAAGA GACAGGATGA GG -            #ATCGTTTC   2940                                                                 - - GCATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GA -            #GAGGCTAT   3000                                                                 - - TCGGCTATGA CTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TT -            #CCGGCTGT   3060                                                                 - - CAGCGCAGGG GCGCCCGGTT CTTTTTGTCA AGACCGACCT GTCCGGTGCC CT -            #GAATGAAC   3120                                                                 - - TGCAGGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT TG -            #CGCAGCTG   3180                                                                 - - TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GT -            #GCCGGGGC   3240                                                                 - - AGGATCTCCT GTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GC -            #TGATGCAA   3300                                                                 - - TGCGGCGGCT GCATACGCTT GATCCGGCTA CCTGCCCATT CGACCACCAA GC -            #GAAACATC   3360                                                                 - - GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT GA -            #TCTGGACG   3420                                                                 - - AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG CG -            #CATGCCCG   3480                                                                 - - ACGGCGAGGA TCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC AT -            #GGTGGAAA   3540                                                                 - - ATGGCCGCTT TTCTGGATTC ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CG -            #CTATCAGG   3600                                                                 - - ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG GC -            #TGACCGCT   3660                                                                 - - TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TA -            #TCGCCTTC   3720                                                                 - - TTGACGAGTT CTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CG -            #ACGCCCAA   3780                                                                 - - CCTGCCATCA CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GC -            #TTCGGAAT   3840                                                                 - - CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TG -            #GAGTTCTT   3900                                                                 - - CGCCCACCCC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA AT -            #AGCATCAC   3960                                                                 - - AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT CC -            #AAACTCAT   4020                                                                 - - CAATGTATCT TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG CG -            #TAATCATG   4080                                                                 - - GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA AC -            #ATACGAGC   4140                                                                 - - CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CA -            #TTAATTGC   4200                                                                 - - GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC AT -            #TAATGAAT   4260                                                                 - - CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CC -            #TCGCTCAC   4320                                                                 - - TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CA -            #AAGGCGGT   4380                                                                 - - AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CA -            #AAAGGCCA   4440                                                                 - - GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GG -            #CTCCGCCC   4500                                                                 - - CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CG -            #ACAGGACT   4560                                                                 - - ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TT -            #CCGACCCT   4620                                                                 - - GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TT -            #TCTCAATG   4680                                                                 - - CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GC -            #TGTGTGCA   4740                                                                 - - CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TT -            #GAGTCCAA   4800                                                                 - - CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TT -            #AGCAGAGC   4860                                                                 - - GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GC -            #TACACTAG   4920                                                                 - - AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AA -            #AGAGTTGG   4980                                                                 - - TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TT -            #TGCAAGCA   5040                                                                 - - GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CT -            #ACGGGGTC   5100                                                                 - - TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TA -            #TCAAAAAG   5160                                                                 - - GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AA -            #AGTATATA   5220                                                                 - - TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TC -            #TCAGCGAT   5280                                                                 - - CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CT -            #ACGATACG   5340                                                                 - - GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GC -            #TCACCGGC   5400                                                                 - - TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GT -            #GGTCCTGC   5460                                                                 - - AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TA -            #AGTAGTTC   5520                                                                 - - GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TG -            #TCACGCTC   5580                                                                 - - GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TT -            #ACATGATC   5640                                                                 - - CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TC -            #AGAAGTAA   5700                                                                 - - GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TT -            #ACTGTCAT   5760                                                                 - - GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TC -            #TGAGAATA   5820                                                                 - - GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CC -            #GCGCCACA   5880                                                                 - - TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AA -            #CTCTCAAG   5940                                                                 - - GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA AC -            #TGATCTTC   6000                                                                 - - AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AA -            #AATGCCGC   6060                                                                 - - AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TT -            #TTTCAATA   6120                                                                 - - TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AA -            #TGTATTTA   6180                                                                 - - GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CT -            #GACGTC     6238                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3699 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..3699                                                         (D) OTHER INFORMATION: - #/note= "pBSGFP"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AA -            #ATCAGCTC     60                                                                 - - ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AA -            #TAGACCGA    120                                                                 - - GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA AC -            #GTGGACTC    180                                                                 - - CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AA -            #CCATCACC    240                                                                 - - CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CT -            #AAAGGGAG    300                                                                 - - CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AA -            #GGGAAGAA    360                                                                 - - AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GC -            #GTAACCAC    420                                                                 - - CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCG CGCCATTCGC CA -            #TTCAGGCT    480                                                                 - - GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG CTATTACGCC AG -            #CTGGCGAA    540                                                                 - - AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTCCC AG -            #TCACGACG    600                                                                 - - TTGTAAAACG ACGGCCAGTG AATTGTAATA CGACTCACTA TAGGGCGAAT TG -            #GGTACCGG    660                                                                 - - GCCCCCCCTC GAGGTCGACG GTATCGATAA GCTTGATGAT CCTTATTTGT AT -            #AGTTCATC    720                                                                 - - CATGCCATGT GTAATCCCAG CAGCTGTTAC AAACTCAAGA AGGACCATGT GG -            #TCTCTCTT    780                                                                 - - TTCGTTGGGA TCTTTCGAAA GGGCAGATTG TGTGGACAGG TAATGGTTGT CT -            #GGTAAAAG    840                                                                 - - GACAGGGCCA TCGCCAATTG GAGTATTTTG TTGATAATGG TCTGCTAGTT GA -            #ACGCTTCC    900                                                                 - - ATCTTCAATG TTGTGTCTAA TTTTGAAGTT AACTTTGATT CCATTCTTTT GT -            #TTGTCTGC    960                                                                 - - CATGATGTAT ACATTGTGTG AGTTATAGTT GTATTCCAAT TTGTGTCCAA GA -            #ATGTTTCC   1020                                                                 - - ATCTTCTTTA AAATCAATAC CTTTTAACTC GATTCTATTA ACAAGGGTAT CA -            #CCTTCAAA   1080                                                                 - - CTTGACTTCA GCACGTGTCT TGTAGTTCCC GTCATCTTTG AAAAATATAG TT -            #CTTTCCTG   1140                                                                 - - TACATAACCT TCGGGCATGG CACTCTTGAA AAAGTCATGC CGTTTCATAT GA -            #TCCGGGTA   1200                                                                 - - TCTTGAAAAG CATTGAACAC CATAAGAGAA AGTAGTGACA AGTGTTGGCC AT -            #GGAACAGG   1260                                                                 - - TAGTTTTCCA GTAGTGCAAA TAAATTTAAG GGTAAGTTTT CCGTATGTTG CA -            #TCACCTTC   1320                                                                 - - ACCCTCTCCA CTGACAGAAA ATTTGTGCCC ATTAACATCA CCATCTAATT CA -            #ACAAGAAT   1380                                                                 - - TGGGACAACT CCAGTGAAGA GTTCTTCTCC TTTGCTAGCC ATTTCTTGCG CG -            #ATCGAATT   1440                                                                 - - CCTGCAGCCC GGGGGATCCA CTAGTTCTAG AGCGGCCGCC ACCGCGGTGG AG -            #CTCCAGCT   1500                                                                 - - TTTGTTCCCT TTAGTGAGGG TTAATTCCGA GCTTGGCGTA ATCATGGTCA TA -            #GCTGTTTC   1560                                                                 - - CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA AG -            #CATAAAGT   1620                                                                 - - GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT AATTGCGTTG CG -            #CTCACTGC   1680                                                                 - - CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA ATGAATCGGC CA -            #ACGCGCGG   1740                                                                 - - GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TC -            #GCTGCGCT   1800                                                                 - - CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CG -            #GTTATCCA   1860                                                                 - - CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AA -            #GGCCAGGA   1920                                                                 - - ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GA -            #CGAGCATC   1980                                                                 - - ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AG -            #ATACCAGG   2040                                                                 - - CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CT -            #TACCGGAT   2100                                                                 - - ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CG -            #CTGTAGGT   2160                                                                 - - ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CC -            #CCCCGTTC   2220                                                                 - - AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GT -            #AAGACACG   2280                                                                 - - ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TA -            #TGTAGGCG   2340                                                                 - - GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG AC -            #AGTATTTG   2400                                                                 - - GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TC -            #TTGATCCG   2460                                                                 - - GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG AT -            #TACGCGCA   2520                                                                 - - GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GC -            #TCAGTGGA   2580                                                                 - - ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TT -            #CACCTAGA   2640                                                                 - - TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TA -            #AACTTGGT   2700                                                                 - - CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CT -            #ATTTCGTT   2760                                                                 - - CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GG -            #CTTACCAT   2820                                                                 - - CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GA -            #TTTATCAG   2880                                                                 - - CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TT -            #ATCCGCCT   2940                                                                 - - CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GT -            #TAATAGTT   3000                                                                 - - TGCGCAACGT TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG TT -            #TGGTATGG   3060                                                                 - - CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC AT -            #GTTGTGCA   3120                                                                 - - AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GC -            #CGCAGTGT   3180                                                                 - - TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TC -            #CGTAAGAT   3240                                                                 - - GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT AT -            #GCGGCGAC   3300                                                                 - - CGAGTTGCTC TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC AG -            #AACTTTAA   3360                                                                 - - AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TT -            #ACCGCTGT   3420                                                                 - - TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TC -            #TTTTACTT   3480                                                                 - - TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AA -            #GGGAATAA   3540                                                                 - - GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TG -            #AAGCATTT   3600                                                                 - - ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AA -            #TAAACAAA   3660                                                                 - - TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTG      - #                      - #  3699                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6361 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..6361                                                         (D) OTHER INFORMATION: - #/note= "pFRED13"                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT CA -             #CAGTTAAA     60                                                                 - - TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT GCGCTCATCG TC -            #ATCCTCGG    120                                                                 - - CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CG -            #GGCCTCTT    180                                                                 - - GCGGGATATC CGGATATAGT TCCTCCTTTC AGCAAAAAAC CCCTCAAGAC CC -            #GTTTAGAG    240                                                                 - - GCCCCAAGGG GTTATGCTAG TTATTGCTCA GCGGTGGCAG CAGCCAACTC AG -            #CTTCCTTT    300                                                                 - - CGGGCTTTGT TAGCAGCCGG ATCCTTATTT GTATAGTTCA TCCATGCCAT GT -            #GTAATCCC    360                                                                 - - AGCAGCTGTT ACAAACTCAA GAAGGACCAT GTGGTCTCTC TTTTCGTTGG GA -            #TCTTTCGA    420                                                                 - - AAGGGCAGAT TGTGTGGACA GGTAATGGTT GTCTGGTAAA AGGACAGGGC CA -            #TCGCCAAT    480                                                                 - - TGGAGTATTT TGTTGATAAT GGTCTGCTAG TTGAACGCTT CCATCTTCAA TG -            #TTGTGTCT    540                                                                 - - AATTTTGAAG TTAACTTTGA TTCCATTCTT TTGTTTGTCT GCCATGATGT AT -            #ACATTGTG    600                                                                 - - TGAGTTATAG TTGTATTCCA ATTTGTGTCC AAGAATGTTT CCATCTTCTT TA -            #AAATCAAT    660                                                                 - - ACCTTTTAAC TCGATTCTAT TAACAAGGGT ATCACCTTCA AACTTGACTT CA -            #GCACGTGT    720                                                                 - - CTTGTAGTTC CCGTCATCTT TGAAAAATAT AGTTCTTTCC TGTACATAAC CT -            #TCGGGCAT    780                                                                 - - GGCACTCTTG AAAAAGTCAT GCCGTTTCAT ATGATCCGGG TATCTTGAAA AG -            #CATTGAAC    840                                                                 - - ACCATAAGAG AAAGTAGTGA CAAGTGTTGG CCATGGAACA GGTAGTTTTC CA -            #GTAGTGCA    900                                                                 - - AATAAATTTA AGGGTAAGTT TTCCGTATGT TGCATCACCT TCACCCTCTC CA -            #CTGACAGA    960                                                                 - - AAATTTGTGC CCATTAACAT CACCATCTAA TTCAACAAGA ATTGGGACAA CT -            #CCAGTGAA   1020                                                                 - - GAGTTCTTCT CCTTTGCTAG CCATATGTAT ATCTCCTTCT TAAAGTTAAA CA -            #AAATTATT   1080                                                                 - - TCTAGAGGGG AATTGTTATC CGCTCACAAT TCCCCTATAG TGAGTCGTAT TA -            #ATTTCGCG   1140                                                                 - - GGATCGAGAT CTCGATCCTC TACGCCGGAC GCATCGTGGC CGGCATCACC GG -            #CGCCACAG   1200                                                                 - - GTGCGGTTGC TGGCGCCTAT ATCGCCGACA TCACCGATGG GGAAGATCGG GC -            #TCGCCACT   1260                                                                 - - TCGGGCTCAT GAGCGCTTGT TTCGGCGTGG GTATGGTGGC AGGCCCCGTG GC -            #CGGGGGAC   1320                                                                 - - TGTTGGGCGC CATCTCCTTG CATGCACCAT TCCTTGCGGC GGCGGTGCTC AA -            #CGGCCTCA   1380                                                                 - - ACCTACTACT GGGCTGCTTC CTAATGCAGG AGTCGCATAA GGGAGAGCGT CG -            #AGATCCCG   1440                                                                 - - GACACCATCG AATGGCGCAA AACCTTTCGC GGTATGGCAT GATAGCGCCC GG -            #AAGAGAGT   1500                                                                 - - CAATTCAGGG TGGTGAATGT GAAACCAGTA ACGTTATACG ATGTCGCAGA GT -            #ATGCCGGT   1560                                                                 - - GTCTCTTATC AGACCGTTTC CCGCGTGGTG AACCAGGCCA GCCACGTTTC TG -            #CGAAAACG   1620                                                                 - - CGGGAAAAAG TGGAAGCGGC GATGGCGGAG CTGAATTACA TTCCCAACCG CG -            #TGGCACAA   1680                                                                 - - CAACTGGCGG GCAAACAGTC GTTGCTGATT GGCGTTGCCA CCTCCAGTCT GG -            #CCCTGCAC   1740                                                                 - - GCGCCGTCGC AAATTGTCGC GGCGATTAAA TCTCGCGCCG ATCAACTGGG TG -            #CCAGCGTG   1800                                                                 - - GTGGTGTCGA TGGTAGAACG AAGCGGCGTC GAAGCCTGTA AAGCGGCGGT GC -            #ACAATCTT   1860                                                                 - - CTCGCGCAAC GCGTCAGTGG GCTGATCATT AACTATCCGC TGGATGACCA GG -            #ATGCCATT   1920                                                                 - - GCTGTGGAAG CTGCCTGCAC TAATGTTCCG GCGTTATTTC TTGATGTCTC TG -            #ACCAGACA   1980                                                                 - - CCCATCAACA GTATTATTTT CTCCCATGAA GACGGTACGC GACTGGGCGT GG -            #AGCATCTG   2040                                                                 - - GTCGCATTGG GTCACCAGCA AATCGCGCTG TTAGCGGGCC CATTAAGTTC TG -            #TCTCGGCG   2100                                                                 - - CGTCTGCGTC TGGCTGGCTG GCATAAATAT CTCACTCGCA ATCAAATTCA GC -            #CGATAGCG   2160                                                                 - - GAACGGGAAG GCGACTGGAG TGCCATGTCC GGTTTTCAAC AAACCATGCA AA -            #TGCTGAAT   2220                                                                 - - GAGGGCATCG TTCCCACTGC GATGCTGGTT GCCAACGATC AGATGGCGCT GG -            #GCGCAATG   2280                                                                 - - CGCGCCATTA CCGAGTCCGG GCTGCGCGTT GGTGCGGATA TCTCGGTAGT GG -            #GATACGAC   2340                                                                 - - GATACCGAAG ACAGCTCATG TTATATCCCG CCGTTAACCA CCATCAAACA GG -            #ATTTTCGC   2400                                                                 - - CTGCTGGGGC AAACCAGCGT GGACCGCTTG CTGCAACTCT CTCAGGGCCA GG -            #CGGTGAAG   2460                                                                 - - GGCAATCAGC TGTTGCCCGT CTCACTGGTG AAAAGAAAAA CCACCCTGGC GC -            #CCAATACG   2520                                                                 - - CAAACCGCCT CTCCCCGCGC GTTGGCCGAT TCATTAATGC AGCTGGCACG AC -            #AGGTTTCC   2580                                                                 - - CGACTGGAAA GCGGGCAGTG AGCGCAACGC AATTAATGTA AGTTAGCTCA CT -            #CATTAGGC   2640                                                                 - - ACCGGGATCT CGACCGATGC CCTTGAGAGC CTTCAACCCA GTCAGCTCCT TC -            #CGGTGGGC   2700                                                                 - - GCGGGGCATG ACTATCGTCG CCGCACTTAT GACTGTCTTC TTTATCATGC AA -            #CTCGTAGG   2760                                                                 - - ACAGGTGCCG GCAGCGCTCT GGGTCATTTT CGGCGAGGAC CGCTTTCGCT GG -            #AGCGCGAC   2820                                                                 - - GATGATCGGC CTGTCGCTTG CGGTATTCGG AATCTTGCAC GCCCTCGCTC AA -            #GCCTTCGT   2880                                                                 - - CACTGGTCCC GCCACCAAAC GTTTCGGCGA GAAGCAGGCC ATTATCGCCG GC -            #ATGGCGGC   2940                                                                 - - CGACGCGCTG GGCTACGTCT TGCTGGCGTT CGCGACGCGA GGCTGGATGG CC -            #TTCCCCAT   3000                                                                 - - TATGATTCTT CTCGCTTCCG GCGGCATCGG GATGCCCGCG TTGCAGGCCA TG -            #CTGTCCAG   3060                                                                 - - GCAGGTAGAT GACGACCATC AGGGACAGCT TCAAGGATCG CTCGCGGCTC TT -            #ACCAGCCT   3120                                                                 - - AACTTCGATC ACTGGACCGC TGATCGTCAC GGCGATTTAT GCCGCCTCGG CG -            #AGCACATG   3180                                                                 - - GAACGGGTTG GCATGGATTG TAGGCGCCGC CCTATACCTT GTCTGCCTCC CC -            #GCGTTGCG   3240                                                                 - - TCGCGGTGCA TGGAGCCGGG CCACCTCGAC CTGAATGGAA GCCGGCGGCA CC -            #TCGCTAAC   3300                                                                 - - GGATTCACCA CTCCAAGAAT TGGAGCCAAT CAATTCTTGC GGAGAACTGT GA -            #ATGCGCAA   3360                                                                 - - ACCAACCCTT GGCAGAACAT ATCCATCGCG TCCGCCATCT CCAGCAGCCG CA -            #CGCGGCGC   3420                                                                 - - ATCTCGGGCA GCGTTGGGTC CTGGCCACGG GTGCGCATGA TCGTGCTCCT GT -            #CGTTGAGG   3480                                                                 - - ACCCGGCTAG GCTGGCGGGG TTGCCTTACT GGTTAGCAGA ATGAATCACC GA -            #TACGCGAG   3540                                                                 - - CGAACGTGAA GCGACTGCTG CTGCAAAACG TCTGCGACCT GAGCAACAAC AT -            #GAATGGTC   3600                                                                 - - TTCGGTTTCC GTGTTTCGTA AAGTCTGGAA ACGCGGAAGT CAGCGCCCTG CA -            #CCATTATG   3660                                                                 - - TTCCGGATCT GCATCGCAGG ATGCTGCTGG CTACCCTGTG GAACACCTAC AT -            #CTGTATTA   3720                                                                 - - ACGAAGCGCT GGCATTGACC CTGAGTGATT TTTCTCTGGT CCCGCCGCAT CC -            #ATACCGCC   3780                                                                 - - AGTTGTTTAC CCTCACAACG TTCCAGTAAC CGGGCATGTT CATCATCAGT AA -            #CCCGTATC   3840                                                                 - - GTGAGCATCC TCTCTCGTTT CATCGGTATC ATTACCCCCA TGAACAGAAA TC -            #CCCCTTAC   3900                                                                 - - ACGGAGGCAT CAGTGACCAA ACAGGAAAAA ACCGCCCTTA ACATGGCCCG CT -            #TTATCAGA   3960                                                                 - - AGCCAGACAT TAACGCTTCT GGAGAAACTC AACGAGCTGG ACGCGGATGA AC -            #AGGCAGAC   4020                                                                 - - ATCTGTGAAT CGCTTCACGA CCACGCTGAT GAGCTTTACC GCAGCTGCCT CG -            #CGCGTTTC   4080                                                                 - - GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AG -            #CTTGTCTG   4140                                                                 - - TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TG -            #GCGGGTGT   4200                                                                 - - CGGGGCGCAG CCATGACCCA GTCACGTAGC GATAGCGGAG TGTATACTGG CT -            #TAACTATG   4260                                                                 - - CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATATG CGGTGTGAAA TA -            #CCGCACAG   4320                                                                 - - ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT TCCTCGCTCA CT -            #GACTCGCT   4380                                                                 - - GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TA -            #ATACGGTT   4440                                                                 - - ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AG -            #CAAAAGGC   4500                                                                 - - CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CC -            #CCTGACGA   4560                                                                 - - GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TA -            #TAAAGATA   4620                                                                 - - CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TG -            #CCGCTTAC   4680                                                                 - - CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GC -            #TCACGCTG   4740                                                                 - - TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC AC -            #GAACCCCC   4800                                                                 - - CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA AC -            #CCGGTAAG   4860                                                                 - - ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CG -            #AGGTATGT   4920                                                                 - - AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GA -            #AGGACAGT   4980                                                                 - - ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GT -            #AGCTCTTG   5040                                                                 - - ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AG -            #CAGATTAC   5100                                                                 - - GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CT -            #GACGCTCA   5160                                                                 - - GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GG -            #ATCTTCAC   5220                                                                 - - CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT AT -            #GAGTAAAC   5280                                                                 - - TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TC -            #TGTCTATT   5340                                                                 - - TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GG -            #GAGGGCTT   5400                                                                 - - ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CT -            #CCAGATTT   5460                                                                 - - ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CA -            #ACTTTATC   5520                                                                 - - CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CG -            #CCAGTTAA   5580                                                                 - - TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG GTGTCACGCT CG -            #TCGTTTGG   5640                                                                 - - TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CC -            #CCCATGTT   5700                                                                 - - GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AG -            #TTGGCCGC   5760                                                                 - - AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TG -            #CCATCCGT   5820                                                                 - - AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AG -            #TGTATGCG   5880                                                                 - - GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC AT -            #AGCAGAAC   5940                                                                 - - TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GG -            #ATCTTACC   6000                                                                 - - GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CA -            #GCATCTTT   6060                                                                 - - TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CA -            #AAAAAGGG   6120                                                                 - - AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT AT -            #TATTGAAG   6180                                                                 - - CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AG -            #AAAAATAA   6240                                                                 - - ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT AA -            #GAAACCAT   6300                                                                 - - TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GT -            #CTTCAAGA   6360                                                                 - - A                  - #                  - #                  - #                 6361                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..48                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17422"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CAATTTGTGT CCCAGAATGT TGCCATCTTC CTTGAAGTCA ATACCTTT  - #                    48                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..47                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17423"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GTCTTGTAGT TGCCGTCATC TTTGAAGAAG ATGCTCCTTT CCTGTAC   - #                    47                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..52                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17424"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CATGGAACAG GCAGTTTGCC AGTAGTGCAG ATGAACTTCA GGGTAAGTTT TC - #                 52                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17425"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CTCCACTGAC AGAGAACTTG TGGCCGTTAA CATCACCATC     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..47                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17426"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CCATCTTCAA TGTTGTGGCG GGTCTTGAAG TTCACTTTGA TTCCATT   - #                    47                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..41                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #17465"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - CGATAAGCTT GAGGATCCTC AGTTGTACAG TTCATCCATG C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 849 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..849                                                          (D) OTHER INFORMATION: - #/note= "pBSGFPsg11"                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - ATGACCATGA TTACGCCAAG CTCGGAATTA ACCCTCACTA AAGGGAACAA AA -             #GCTGGAGC     60                                                                 - - TCCACCGCGG TGGCGGCCGC TCTAGAACTA GTGGATCCCC CGGGCTGCAG GA -            #ATTCGATC    120                                                                 - - GCGCAAGAAA TGGCTAGCAA AGGAGAAGAA CTCTTCACTG GAGTTGTCCC AA -            #TTCTTGTT    180                                                                 - - GAATTAGATG GTGATGTTAA CGGCCACAAG TTCTCTGTCA GTGGAGAGGG TG -            #AAGGTGAT    240                                                                 - - GCAACATACG GAAAACTTAC CCTGAAGTTC ATCTGCACTA CTGGCAAACT GC -            #CTGTTCCA    300                                                                 - - TGGCCAACAC TTGTCACTAC TCTCTCTTAT GGTGTTCAAT GCTTTTCAAG AT -            #ACCCGGAT    360                                                                 - - CATATGAAAC GGCATGACTT TTTCAAGAGT GCCATGCCCG AAGGTTATGT AC -            #AGGAAAGG    420                                                                 - - ACCATCTTCT TCAAAGATGA CGGCAACTAC AAGACACGTG CTGAAGTCAA GT -            #TTGAAGGT    480                                                                 - - GATACCCTTG TTAATAGAAT CGAGTTAAAA GGTATTGACT TCAAGGAAGA TG -            #GCAACATT    540                                                                 - - CTGGGACACA AATTGGAATA CAACTATAAC TCACACAATG TATACATCAT GG -            #CAGACAAA    600                                                                 - - CAAAAGAATG GAATCAAAGT GAACTTCAAG ACCCGCCACA ACATTGAAGA TG -            #GAAGCGTT    660                                                                 - - CAACTAGCAG ACCATTATCA ACAAAATACT CCAATTGGCG ATGGCCCTGT CC -            #TTTTACCA    720                                                                 - - GACAACCATT ACCTGTCCAC ACAATCTGCC CTTTCGAAAG ATCCCAACGA AA -            #AGAGAGAC    780                                                                 - - CACATGGTCC TTCTTGAGTT TGTAACAGCT GCTGGGATTA CACATGGCAT GG -            #ATGAACTG    840                                                                 - - TACAACTGA                - #                  - #                      - #        849                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SG12"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -             #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTTGTCACTA CTCTCTCTTA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAT TTTAAAGAAG ATGGAAACAT TC -            #TTGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TTAACTTCAA AATTAGACAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT AT -            #ACAAATAA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SG11"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -            #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTTGTCACTA CTCTCTCTTA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAC TTCAAGGAAG ATGGCAACAT TC -            #TGGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TGAACTTCAA GACCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT GT -            #ACAACTGA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SG25"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -            #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTAGTCACTA CTCTGTGCTA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAC TTCAAGGAAG ATGGCAACAT TC -            #TGGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TGAACTTCAA GACCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT GT -            #ACAACTGA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #18217"           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - CATTGAACAC CATAGCACAG AGTAGTGACT AGTGTTGGCC     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SB42"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -             #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTAGTCACTA CTCTCTCTCA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAT TTTAAAGAAG ATGGAAACAT TC -            #TTGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TTAACTTCAA AATTAGACAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT AT -            #ACAAATAA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #bio25"           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CATTGAACAC CATGAGAGAG AGTAGTGACT AGTGTTGGCC     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SB49"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -             #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTAGTCACTA CTTTCTCTCA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAT TTTAAAGAAG ATGGAAACAT TC -            #TTGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG CGAACTTCAA GATCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT AT -            #ACAAATAA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..44                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #19059"           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CTTCAATGTT GTGGCGGATC TTGAAGTTCG CTTTGATTCC ATTC   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..40                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #bio24"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - CATTGAACAC CATGAGAGAA AGTAGTGACT AGTGTTGGCC     - #                      - #    40                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..720                                                          (D) OTHER INFORMATION: - #/note= "SB50"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -             #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTAGTCACTA CTCTCTCTCA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAT TTTAAAGAAG ATGGAAACAT TC -            #TTGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG CGAACTTCAA GATCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT AT -            #ACAAATAA    720                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1521 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..1521                                                         (D) OTHER INFORMATION: - #/note= "pCMVgfo11"                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -            #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTTGTCACTA CTCTCTCTTA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAC TTCAAGGAAG ATGGCAACAT TC -            #TGGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TGAACTTCAA GACCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT GT -            #ACAACGGT    720                                                                 - - GCTGGTGCTA TCGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GG -            #TGGAGAGG    780                                                                 - - CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CG -            #TGTTCCGG    840                                                                 - - CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TG -            #CCCTGAAT    900                                                                 - - GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT TC -            #CTTGCGCA    960                                                                 - - GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG CG -            #AAGTGCCG   1020                                                                 - - GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CA -            #TGGCTGAT   1080                                                                 - - GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CC -            #AAGCGAAA   1140                                                                 - - CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GG -            #ATGATCTG   1200                                                                 - - GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GG -            #CGCGCATG   1260                                                                 - - CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA TA -            #TCATGGTG   1320                                                                 - - GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GG -            #ACCGCTAT   1380                                                                 - - CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA AT -            #GGGCTGAC   1440                                                                 - - CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CT -            #TCTATCGC   1500                                                                 - - CTTCTTGACG AGTTCTTCTG A           - #                  - #                    1521                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Gly Ala Gly Ala                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..32                                                           (D) OTHER INFORMATION: - #/note= "primer Bio51"                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - CGCGGATCCT TCGAACAAGA TGGATTGCAC GC       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..34                                                           (D) OTHER INFORMATION: - #/note= "primer Bio52"                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - CCGGAATTCT CAGAAGAACT CGTCAAGAAG GCGA       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..46                                                           (D) OTHER INFORMATION: - #/note= "primer Bio49"                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - GGCGCGCAAG AAATGGCTAG CAAAGGAGAA GAACTCTTCA CTGGAG   - #                     46                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..46                                                           (D) OTHER INFORMATION: - #/note= "primer Bio50"                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - CCCATCGATA GCACCAGCAC CGTTGTACAG TTCATCCATG CCATGT   - #                     46                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1521 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..1521                                                         (D) OTHER INFORMATION: - #/note= "pPGKgfo25"                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - ATGGCTAGCA AAGGAGAAGA ACTCTTCACT GGAGTTGTCC CAATTCTTGT TG -             #AATTAGAT     60                                                                 - - GGTGATGTTA ACGGCCACAA GTTCTCTGTC AGTGGAGAGG GTGAAGGTGA TG -            #CAACATAC    120                                                                 - - GGAAAACTTA CCCTGAAGTT CATCTGCACT ACTGGCAAAC TGCCTGTTCC AT -            #GGCCAACA    180                                                                 - - CTAGTCACTA CTCTGTGCTA TGGTGTTCAA TGCTTTTCAA GATACCCGGA TC -            #ATATGAAA    240                                                                 - - CGGCATGACT TTTTCAAGAG TGCCATGCCC GAAGGTTATG TACAGGAAAG GA -            #CCATCTTC    300                                                                 - - TTCAAAGATG ACGGCAACTA CAAGACACGT GCTGAAGTCA AGTTTGAAGG TG -            #ATACCCTT    360                                                                 - - GTTAATAGAA TCGAGTTAAA AGGTATTGAC TTCAAGGAAG ATGGCAACAT TC -            #TGGGACAC    420                                                                 - - AAATTGGAAT ACAACTATAA CTCACACAAT GTATACATCA TGGCAGACAA AC -            #AAAAGAAT    480                                                                 - - GGAATCAAAG TGAACTTCAA GACCCGCCAC AACATTGAAG ATGGAAGCGT TC -            #AACTAGCA    540                                                                 - - GACCATTATC AACAAAATAC TCCAATTGGC GATGGCCCTG TCCTTTTACC AG -            #ACAACCAT    600                                                                 - - TACCTGTCCA CACAATCTGC CCTTTCGAAA GATCCCAACG AAAAGAGAGA CC -            #ACATGGTC    660                                                                 - - CTTCTTGAGT TTGTAACAGC TGCTGGGATT ACACATGGCA TGGATGAACT GT -            #ACAACGGT    720                                                                 - - GCTGGTGCTA TCGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GG -            #TGGAGAGG    780                                                                 - - CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CG -            #TGTTCCGG    840                                                                 - - CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TG -            #CCCTGAAT    900                                                                 - - GAACTGCAGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT TC -            #CTTGCGCA    960                                                                 - - GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG CG -            #AAGTGCCG   1020                                                                 - - GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CA -            #TGGCTGAT   1080                                                                 - - GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CC -            #AAGCGAAA   1140                                                                 - - CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GG -            #ATGATCTG   1200                                                                 - - GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GG -            #CGCGCATG   1260                                                                 - - CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA TA -            #TCATGGTG   1320                                                                 - - GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GG -            #ACCGCTAT   1380                                                                 - - CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA AT -            #GGGCTGAC   1440                                                                 - - CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CT -            #TCTATCGC   1500                                                                 - - CTTCTTGACG AGTTCTTCTG A           - #                  - #                    1521                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #18990"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - GACCGGGACA CGTATCCAGC CTCCGC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: - #/note= "oligonucleotide #18991"            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - GGAGGCTGGA TACGTGTCCC GGTCTGCA         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7617 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..7617                                                         (D) OTHER INFORMATION: - #/note= "pGen-PGKgfo25RO"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - TCGAGGTCGA CGGTATCGAT TAGTCCAATT TGTTAAAGAC AGGATATCAG TG -             #GTCCAGGC     60                                                                 - - TCTAGTTTTG ACTCAACAAT ATCACCAGCT GAAGCCTATA GAGTACGAGC CA -            #TAGATAAA    120                                                                 - - ATAAAAGATT TTATTTAGTC TCCAGAAAAA GGGGGGAATG AAAGACCCCA CC -            #TGTAGGTT    180                                                                 - - TGGCAAGCTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG AAAAATACAT AA -            #CTGAGAAT    240                                                                 - - AGAGAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT GAATATGGGC CA -            #AACAGGAT    300                                                                 - - ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA ACAGATGGAA CA -            #GCTGAATA    360                                                                 - - TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC GGCTCAGGGC CA -            #AGAACAGA    420                                                                 - - TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA GTTTCTAGAG AACCATCAGA TG -            #TTTCCAGG    480                                                                 - - GTGCCCCAAG GACCTGAAAT GACCCTGTGC CTTATTTGAA CTAACCAATC AG -            #TTCGCTTC    540                                                                 - - TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA AAGAGCCCAC AA -            #CCCCTCAC    600                                                                 - - TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA CCCGTGTATC CA -            #ATAAACCC    660                                                                 - - TCTTGCAGTT GCATCCGACT TGTGGTCTCG CTGTTCCTTG GGAGGGTCTC CT -            #CTGAGTGA    720                                                                 - - TTGACTACCC GTCAGCGGGG GTCTTTCATT TGGGGGCTCG TCCGGGATCG GG -            #AGACCCCT    780                                                                 - - GCCCAGGGAC CACCGACCCA CCACCGGGAG GTAAGCTGGC CAGCAACTTA TC -            #TGTGTCTG    840                                                                 - - TCCGATTGTC TAGTGTCTAT GACTGATTTT ATGCGCCTGC GTCGGTACTA GT -            #TAGCTAAC    900                                                                 - - TAGCTCTGTA TCTGGCGGAC CCGTGGTGGA ACTGACGAGT TCGGAACACC CG -            #GCCGCAAC    960                                                                 - - CCTGGGAGAC GTCCCAGGGA CTTCGGGGGC CGTTTTTGTG GCCCGACCTG AG -            #TCCAAAAA   1020                                                                 - - TCCCGATCGT TTTGGACTCT TTGGTGCACC CCCCTTAGAG GAGGGATATG TG -            #GTTCTGGT   1080                                                                 - - AGGAGACGAG AACCTAAAAC AGTTCCCGCC TCCGTCTGAA TTTTTGCTTT CG -            #GTTTGGGA   1140                                                                 - - CCGAAGCCGC GCCGCGCGTC TTGTCTGCTG CAGCATCGTT CTGTGTTGTC TC -            #TGTCTGAC   1200                                                                 - - TGTGTTTCTG TATTTGTCTG AGAATATGGG CCAGACTGTT ACCACTCCCT TA -            #AGTTTGAC   1260                                                                 - - CTTAGGTCAC TGGAAAGATG TCGAGCGGAT CGCTCACAAC CAGTCGGTAG AT -            #GTCAAGAA   1320                                                                 - - GAGACGTTGG GTTACCTTCT GCTCTGCAGA ATGGCCAACC TTTAACGTCG GA -            #TGGCCGCG   1380                                                                 - - AGACGGCACC TTTAACCGAG ACCTCATCAC CCAGGTTAAG ATCAAGGTCT TT -            #TCACCTGG   1440                                                                 - - CCCGCATGGA CACCCAGACC AGGTCCCCTA CATCGTGACC TGGGAAGCCT TG -            #GCTTTTGA   1500                                                                 - - CCCCCCTCCC TGGGTCAAGC CCTTTGTACA CCCTAAGCCT CCGCCTCCTC TT -            #CCTCCATC   1560                                                                 - - CGCCCCGTCT CTCCCCCTTG AACCTCCTCG TTCGACCCCG CCTCGATCCT CC -            #CTTTATCC   1620                                                                 - - AGCCCTCACT CCTTCTCGAC GGTATACAGA CATGATAAGA TACATTGATG AG -            #TTTGGACA   1680                                                                 - - AACCACAACT AGAATGCAGT GAAAAAAATG CTTTATTTGT GAAATTTGTG AT -            #GCTATTGC   1740                                                                 - - TTTATTTGTA ACCATTATAA GCTGCAATAA ACAAGTTGGG GTGGGCGAAG AA -            #CTCCAGCA   1800                                                                 - - TGAGATCCCC GCGCTGGAGG ATCATCCAGC CGGCGAACGT GGCGAGAAAG GA -            #AGGGAAGA   1860                                                                 - - AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC GGTCACGCTG CG -            #CGTAACCA   1920                                                                 - - CCACACCCGC CGCGCTTAAT GCGCCGCTAC AGGGCGCGTG GGGATACCCC CT -            #AGAGCCCC   1980                                                                 - - AGCTGGTTCT TTCCGCCTCA GAAGCCATAG AGCCCACCGC ATCCCCAGCA TG -            #CCTGCTAT   2040                                                                 - - TGTCTTCCCA ATCCTCCCCC TTGCTGTCCT GCCCCACCCC ACCCCCCAGA AT -            #AGAATGAC   2100                                                                 - - ACCTACTCAG ACAATGCGAT GCAATTTCCT CATTTTATTA GGAAAGGACA GT -            #GGGAGTGG   2160                                                                 - - CACCTTCCAG GGTCAAGGAA GGCACGGGGG AGGGGCAAAC AACAGATGGC TG -            #GCAACTAG   2220                                                                 - - AAGGCACAGT CGAGGCTGAT CAGCGAGCTC TAGCATTTAG GTGACACTAT AG -            #AATAGGGC   2280                                                                 - - CCTCTAGATG CATGCTCGAG CGGCCGCCAG TGTGATGGAT ATCTGCAGAA TT -            #CTCAGAAG   2340                                                                 - - AACTCGTCAA GAAGGCGATA GAAGGCGATG CGCTGCGAAT CGGGAGCGGC GA -            #TACCGTAA   2400                                                                 - - AGCACGAGGA AGCGGTCAGC CCATTCGCCG CCAAGCTCTT CAGCAATATC AC -            #GGGTAGCC   2460                                                                 - - AACGCTATGT CCTGATAGCG GTCCGCCACA CCCAGCCGGC CACAGTCGAT GA -            #ATCCAGAA   2520                                                                 - - AAGCGGCCAT TTTCCACCAT GATATTCGGC AAGCAGGCAT CGCCATGGGT CA -            #CGACGAGA   2580                                                                 - - TCCTCGCCGT CGGGCATGCG CGCCTTGAGC CTGGCGAACA GTTCGGCTGG CG -            #CGAGCCCC   2640                                                                 - - TGATGCTCTT CGTCCAGATC ATCCTGATCG ACAAGACCGG CTTCCATCCG AG -            #TACGTGCT   2700                                                                 - - CGCTCGATGC GATGTTTCGC TTGGTGGTCG AATGGGCAGG TAGCCGGATC AA -            #GCGTATGC   2760                                                                 - - AGCCGCCGCA TTGCATCAGC CATGATGGAT ACTTTCTCGG CAGGAGCAAG GT -            #GAGATGAC   2820                                                                 - - AGGAGATCCT GCCCCGGCAC TTCGCCCAAT AGCAGCCAGT CCCTTCCCGC TT -            #CAGTGACA   2880                                                                 - - ACGTCGAGCA CAGCTGCGCA AGGAACGCCC GTCGTGGCCA GCCACGATAG CC -            #GCGCTGCC   2940                                                                 - - TCGTCCTGCA GTTCATTCAG GGCACCGGAC AGGTCGGTCT TGACAAAAAG AA -            #CCGGGCGC   3000                                                                 - - CCCTGCGCTG ACAGCCGGAA CACGGCGGCA TCAGAGCAGC CGATTGTCTG TT -            #GTGCCCAG   3060                                                                 - - TCATAGCCGA ATAGCCTCTC CACCCAAGCG GCCGGAGAAC CTGCGTGCAA TC -            #CATCTTGT   3120                                                                 - - TCGATAGCAC CAGCACCGTT GTACAGTTCA TCCATGCCAT GTGTAATCCC AG -            #CAGCTGTT   3180                                                                 - - ACAAACTCAA GAAGGACCAT GTGGTCTCTC TTTTCGTTGG GATCTTTCGA AA -            #GGGCAGAT   3240                                                                 - - TGTGTGGACA GGTAATGGTT GTCTGGTAAA AGGACAGGGC CATCGCCAAT TG -            #GAGTATTT   3300                                                                 - - TGTTGATAAT GGTCTGCTAG TTGAACGCTT CCATCTTCAA TGTTGTGGCG GG -            #TCTTGAAG   3360                                                                 - - TTCACTTTGA TTCCATTCTT TTGTTTGTCT GCCATGATGT ATACATTGTG TG -            #AGTTATAG   3420                                                                 - - TTGTATTCCA ATTTGTGTCC CAGAATGTTG CCATCTTCCT TGAAGTCAAT AC -            #CTTTTAAC   3480                                                                 - - TCGATTCTAT TAACAAGGGT ATCACCTTCA AACTTGACTT CAGCACGTGT CT -            #TGTAGTTG   3540                                                                 - - CCGTCATCTT TGAAGAAGAT GGTCCTTTCC TGTACATAAC CTTCGGGCAT GG -            #CACTCTTG   3600                                                                 - - AAAAAGTCAT GCCGTTTCAT ATGATCCGGG TATCTTGAAA AGCATTGAAC AC -            #CATAGCAC   3660                                                                 - - AGAGTAGTGA CTAGTGTTGG CCATGGAACA GGCAGTTTGC CAGTAGTGCA GA -            #TGAACTTC   3720                                                                 - - AGGGTAAGTT TTCCGTATGT TGCATCACCT TCACCCTCTC CACTGACAGA GA -            #ACTTGTGG   3780                                                                 - - CCGTTAACAT CACCATCTAA TTCAACAAGA ATTGGGACAA CTCCAGTGAA GA -            #GTTCTTCT   3840                                                                 - - CCTTTGCTAG CCATTTCTTG CGCGCCCGCG GAGGCTGGAT ACGTGTCCCG GT -            #CTGCAGGT   3900                                                                 - - CGAAAGGCCC GGAGATGAGG AAGAGGAGAA CAGCGCGGCA GACGTGCGCT TT -            #TGAAGCGT   3960                                                                 - - GCAGAATGCC GGGCTCCGGA GGACCTTCGC GCCCGCCCCG CCCCTGAGCC CG -            #CCCCTGAG   4020                                                                 - - CCCGCCCCCG GACCCACCCC TTCCCAGCCT CTGAGCCCAG AAAGCGAAGG AG -            #CCAAGCTG   4080                                                                 - - CTATTGGCCG CTGCCCCAAA GGCCTACCCG CTTCCATTGC TCAGCGGTGC TG -            #TCCATCTG   4140                                                                 - - CACGAGACTA GTGAGACGTG CTACTTCCAT TTGTCACGTC CTGCACGACG CG -            #AGCTGCGG   4200                                                                 - - GGCGGGGGGG AACTTCCTGA CTAGGGGAGG AGTAGAAGGT GGCGCGAAGG GG -            #CCACCAAA   4260                                                                 - - GAAGGGAGCC GGTTGGCGCT ACCGGTGGAT GTGGAATGTG TGCGAGGCCA GA -            #GGCCACTT   4320                                                                 - - GTGTAGCGCC AAGTGCCAGC GGGGCTGCTA AAGCGCATGC TCCAGACTGC CT -            #TGGGAAAA   4380                                                                 - - GCGCCTCCCC TACCCGGTAG AATTCGATAT CAAGCTTATC GATACCGTCG AG -            #ATCTCCCG   4440                                                                 - - ATCCGTCGAG GTCGACGGTA TCGATTAGTC CAATTTGTTA AAGACAGGAT AT -            #CAGTGGTC   4500                                                                 - - CAGGCTCTAG TTTTGACTCA ACAATATCAC CAGCTGAAGC CTATAGAGTA CG -            #AGCCATAG   4560                                                                 - - ATAAAATAAA AGATTTTATT TAGTCTCCAG AAAAAGGGGG GAATGAAAGA CC -            #CCACCTGT   4620                                                                 - - AGGTTTGGCA AGCTAGCTTA AGTAACGCCA TTTTGCAAGG CATGGAAAAA TA -            #CATAACTG   4680                                                                 - - AGAATAGAGA AGTTCAGATC GGGATCCCAA TTCTTTCGGA CTTTTGAAAG TG -            #ATGGTGGT   4740                                                                 - - GGGGGAAGGA TTCGAACCTT CGAAGTCGAT GACGGCAGAT TTAGAGTCTG CT -            #CCCTTTGG   4800                                                                 - - CCGCTCGGGA ACCCCACCAC GGGTAATGCT TTTACTGGCC TGCTCCCTTA TC -            #GGGAAGCG   4860                                                                 - - GGGCGCATCA TATCAAATGA CGCGCCGCTG TAAAGTGTTA CGTTGAGAAA GA -            #ATTGGGAT   4920                                                                 - - CCCGATCAAG GTCAGGAACA GATGGAACAG CTAGAGAACC ATCAGATGTT TC -            #CAGGGTGC   4980                                                                 - - CCCAAGGACC TGAAATGACC CTGTGCCTTA TTTGAACTAA CCAATCAGTT CG -            #CTTCTCGC   5040                                                                 - - TTCTGTTCGC GCGCTTCTGC TCCCCGAGCT CAATAAAAGA GCCCACAACC CC -            #TCACTCGG   5100                                                                 - - GGCGCCAGTC CTCCGATTGA CTGAGTCGCC CGGGTACCCG TGTATCCAAT AA -            #ACCCTCTT   5160                                                                 - - GCAGTTGCAT CCGACTTGTG GTCTCGCTGT TCCTTGGGAG GGTCTCCTCT GA -            #GTGATTGA   5220                                                                 - - CTACCCGTCA GCGGGGGTCT TTCACCCAGA GTTTGGAACT TACTGTCTTC TT -            #GGGACCTG   5280                                                                 - - CAGCCCGGGG GATCCACTAG TTCTAGAGCG GCCGCCACCG CGGTGGATTC TG -            #CCTCGCGC   5340                                                                 - - GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GT -            #CACAGCTT   5400                                                                 - - GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG GG -            #TGTTGGCG   5460                                                                 - - GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG CGGAGTGTAT AC -            #TGGCTTAA   5520                                                                 - - CTATGCGGCA TCAGAGCAGA TTGTACTGAG AGTGCACCAT ATGCGGTGTG AA -            #ATACCGCA   5580                                                                 - - CAGATGCGTA AGGAGAAAAT ACCGCATCAG GCGCTCTTCC GCTTCCTCGC TC -            #ACTGACTC   5640                                                                 - - GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CG -            #GTAATACG   5700                                                                 - - GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GC -            #CAGCAAAA   5760                                                                 - - GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GC -            #CCCCCTGA   5820                                                                 - - CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GA -            #CTATAAAG   5880                                                                 - - ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CC -            #CTGCCGCT   5940                                                                 - - TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AA -            #TGCTCACG   6000                                                                 - - CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TG -            #CACGAACC   6060                                                                 - - CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CC -            #AACCCGGT   6120                                                                 - - AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GA -            #GCGAGGTA   6180                                                                 - - TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CT -            #AGAAGGAC   6240                                                                 - - AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TT -            #GGTAGCTC   6300                                                                 - - TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AG -            #CAGCAGAT   6360                                                                 - - TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GG -            #TCTGACGC   6420                                                                 - - TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AA -            #AGGATCTT   6480                                                                 - - CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TA -            #TATGAGTA   6540                                                                 - - AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CG -            #ATCTGTCT   6600                                                                 - - ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TA -            #CGGGAGGG   6660                                                                 - - CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CG -            #GCTCCAGA   6720                                                                 - - TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CT -            #GCAACTTT   6780                                                                 - - ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GT -            #TCGCCAGT   6840                                                                 - - TAATAGTTTG CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GC -            #TCGTCGTT   6900                                                                 - - TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GA -            #TCCCCCAT   6960                                                                 - - GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GT -            #AAGTTGGC   7020                                                                 - - CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TC -            #ATGCCATC   7080                                                                 - - CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AA -            #TAGTGTAT   7140                                                                 - - GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CA -            #CATAGCAG   7200                                                                 - - AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CA -            #AGGATCTT   7260                                                                 - - ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CT -            #TCAGCATC   7320                                                                 - - TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CC -            #GCAAAAAA   7380                                                                 - - GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AA -            #TATTATTG   7440                                                                 - - AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TT -            #TAGAAAAA   7500                                                                 - - TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TC -            #TAAGAAAC   7560                                                                 - - CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TT - #CGTCT          7617                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15581 base - #pairs                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..15581                                                        (D) OTHER INFORMATION: - #/note= "pNLnSG11"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - TGGAAGGGCT AATTTGGTCC CAAAAAAGAC AAGAGATCCT TGATCTGTGG AT -             #CTACCACA     60                                                                 - - CACAAGGCTA CTTCCCTGAT TGGCAGAACT ACACACCAGG GCCAGGGATC AG -            #ATATCCAC    120                                                                 - - TGACCTTTGG ATGGTGCTTC AAGTTAGTAC CAGTTGAACC AGAGCAAGTA GA -            #AGAGGCCA    180                                                                 - - AATAAGGAGA GAAGAACAGC TTGTTACACC CTATGAGCCA GCATGGGATG GA -            #GGACCCGG    240                                                                 - - AGGGAGAAGT ATTAGTGTGG AAGTTTGACA GCCTCCTAGC ATTTCGTCAC AT -            #GGCCCGAG    300                                                                 - - AGCTGCATCC GGAGTACTAC AAAGACTGCT GACATCGAGC TTTCTACAAG GG -            #ACTTTCCG    360                                                                 - - CTGGGGACTT TCCAGGGAGG TGTGGCCTGG GCGGGACTGG GGAGTGGCGA GC -            #CCTCAGAT    420                                                                 - - GCTACATATA AGCAGCTGCT TTTTGCCTGT ACTGGGTCTC TCTGGTTAGA CC -            #AGATCTGA    480                                                                 - - GCCTGGGAGC TCTCTGGCTA ACTAGGGAAC CCACTGCTTA AGCCTCAATA AA -            #GCTTGCCT    540                                                                 - - TGAGTGCTCA AAGTAGTGTG TGCCCGTCTG TTGTGTGACT CTGGTAACTA GA -            #GATCCCTC    600                                                                 - - AGACCCTTTT AGTCAGTGTG GAAAATCTCT AGCAGTGGCG CCCGAACAGG GA -            #CTTGAAAG    660                                                                 - - CGAAAGTAAA GCCAGAGGAG ATCTCTCGAC GCAGGACTCG GCTTGCTGAA GC -            #GCGCACGG    720                                                                 - - CAAGAGGCGA GGGGCGGCGA CTGGTGAGTA CGCCAAAAAT TTTGACTAGC GG -            #AGGCTAGA    780                                                                 - - AGGAGAGAGA TGGGTGCGAG AGCGTCGGTA TTAAGCGGGG GAGAATTAGA TA -            #AATGGGAA    840                                                                 - - AAAATTCGGT TAAGGCCAGG GGGAAAGAAA CAATATAAAC TAAAACATAT AG -            #TATGGGCA    900                                                                 - - AGCAGGGAGC TAGAACGATT CGCAGTTAAT CCTGGCCTTT TAGAGACATC AG -            #AAGGCTGT    960                                                                 - - AGACAAATAC TGGGACAGCT ACAACCATCC CTTCAGACAG GATCAGAAGA AC -            #TTAGATCA   1020                                                                 - - TTATATAATA CAATAGCAGT CCTCTATTGT GTGCATCAAA GGATAGATGT AA -            #AAGACACC   1080                                                                 - - AAGGAAGCCT TAGATAAGAT AGAGGAAGAG CAAAACAAAA GTAAGAAAAA GG -            #CACAGCAA   1140                                                                 - - GCAGCAGCTG ACACAGGAAA CAACAGCCAG GTCAGCCAAA ATTACCCTAT AG -            #TGCAGAAC   1200                                                                 - - CTCCAGGGGC AAATGGTACA TCAGGCCATA TCACCTAGAA CTTTAAATGC AT -            #GGGTAAAA   1260                                                                 - - GTAGTAGAAG AGAAGGCTTT CAGCCCAGAA GTAATACCCA TGTTTTCAGC AT -            #TATCAGAA   1320                                                                 - - GGAGCCACCC CACAAGATTT AAATACCATG CTAAACACAG TGGGGGGACA TC -            #AAGCAGCC   1380                                                                 - - ATGCAAATGT TAAAAGAGAC CATCAATGAG GAAGCTGCAG AATGGGATAG AT -            #TGCATCCA   1440                                                                 - - GTGCATGCAG GGCCTATTGC ACCAGGCCAG ATGAGAGAAC CAAGGGGAAG TG -            #ACATAGCA   1500                                                                 - - GGAACTACTA GTACCCTTCA GGAACAAATA GGATGGATGA CACATAATCC AC -            #CTATCCCA   1560                                                                 - - GTAGGAGAAA TCTATAAAAG ATGGATAATC CTGGGATTAA ATAAAATAGT AA -            #GAATGTAT   1620                                                                 - - AGCCCTACCA GCATTCTGGA CATAAGACAA GGACCAAAGG AACCCTTTAG AG -            #ACTATGTA   1680                                                                 - - GACCGATTCT ATAAAACTCT AAGAGCCGAG CAAGCTTCAC AAGAGGTAAA AA -            #ATTGGATG   1740                                                                 - - ACAGAAACCT TGTTGGTCCA AAATGCGAAC CCAGATTGTA AGACTATTTT AA -            #AAGCATTG   1800                                                                 - - GGACCAGGAG CGACACTAGA AGAAATGATG ACAGCATGTC AGGGAGTGGG GG -            #GACCCGGC   1860                                                                 - - CATAAAGCAA GAGTTTTGGC TGAAGCAATG AGCCAAGTAA CAAATCCAGC TA -            #CCATAATG   1920                                                                 - - ATACAGAAAG GCAATTTTAG GAACCAAAGA AAGACTGTTA AGTGTTTCAA TT -            #GTGGCAAA   1980                                                                 - - GAAGGGCACA TAGCCAAAAA TTGCAGGGCC CCTAGGAAAA AGGGCTGTTG GA -            #AATGTGGA   2040                                                                 - - AAGGAAGGAC ACCAAATGAA AGATTGTACT GAGAGACAGG CTAATTTTTT AG -            #GGAAGATC   2100                                                                 - - TGGCCTTCCC ACAAGGGAAG GCCAGGGAAT TTTCTTCAGA GCAGACCAGA GC -            #CAACAGCC   2160                                                                 - - CCACCAGAAG AGAGCTTCAG GTTTGGGGAA GAGACAACAA CTCCCTCTCA GA -            #AGCAGGAG   2220                                                                 - - CCGATAGACA AGGAACTGTA TCCTTTAGCT TCCCTCAGAT CACTCTTTGG CA -            #GCGACCCC   2280                                                                 - - TCGTCACAAT AAAGATAGGG GGGCAATTAA AGGAAGCTCT ATTAGATACA GG -            #AGCAGATG   2340                                                                 - - ATACAGTATT AGAAGAAATG AATTTGCCAG GAAGATGGAA ACCAAAAATG AT -            #AGGGGGAA   2400                                                                 - - TTGGAGGTTT TATCAAAGTA GGACAGTATG ATCAGATACT CATAGAAATC TG -            #CGGACATA   2460                                                                 - - AAGCTATAGG TACAGTATTA GTAGGACCTA CACCTGTCAA CATAATTGGA AG -            #AAATCTGT   2520                                                                 - - TGACTCAGAT TGGCTGCACT TTAAATTTTC CCATTAGTCC TATTGAGACT GT -            #ACCAGTAA   2580                                                                 - - AATTAAAGCC AGGAATGGAT GGCCCAAAAG TTAAACAATG GCCATTGACA GA -            #AGAAAAAA   2640                                                                 - - TAAAAGCATT AGTAGAAATT TGTACAGAAA TGGAAAAGGA AGGAAAAATT TC -            #AAAAATTG   2700                                                                 - - GGCCTGAAAA TCCATACAAT ACTCCAGTAT TTGCCATAAA GAAAAAAGAC AG -            #TACTAAAT   2760                                                                 - - GGAGAAAATT AGTAGATTTC AGAGAACTTA ATAAGAGAAC TCAAGATTTC TG -            #GGAAGTTC   2820                                                                 - - AATTAGGAAT ACCACATCCT GCAGGGTTAA AACAGAAAAA ATCAGTAACA GT -            #ACTGGATG   2880                                                                 - - TGGGCGATGC ATATTTTTCA GTTCCCTTAG ATAAAGACTT CAGGAAGTAT AC -            #TGCATTTA   2940                                                                 - - CCATACCTAG TATAAACAAT GAGACACCAG GGATTAGATA TCAGTACAAT GT -            #GCTTCCAC   3000                                                                 - - AGGGATGGAA AGGATCACCA GCAATATTCC AGTGTAGCAT GACAAAAATC TT -            #AGAGCCTT   3060                                                                 - - TTAGAAAACA AAATCCAGAC ATAGTCATCT ATCAATACAT GGATGATTTG TA -            #TGTAGGAT   3120                                                                 - - CTGACTTAGA AATAGGGCAG CATAGAACAA AAATAGAGGA ACTGAGACAA CA -            #TCTGTTGA   3180                                                                 - - GGTGGGGATT TACCACACCA GACAAAAAAC ATCAGAAAGA ACCTCCATTC CT -            #TTGGATGG   3240                                                                 - - GTTATGAACT CCATCCTGAT AAATGGACAG TACAGCCTAT AGTGCTGCCA GA -            #AAAGGACA   3300                                                                 - - GCTGGACTGT CAATGACATA CAGAAATTAG TGGGAAAATT GAATTGGGCA AG -            #TCAGATTT   3360                                                                 - - ATGCAGGGAT TAAAGTAAGG CAATTATGTA AACTTCTTAG GGGAACCAAA GC -            #ACTAACAG   3420                                                                 - - AAGTAGTACC ACTAACAGAA GAAGCAGAGC TAGAACTGGC AGAAAACAGG GA -            #GATTCTAA   3480                                                                 - - AAGAACCGGT ACATGGAGTG TATTATGACC CATCAAAAGA CTTAATAGCA GA -            #AATACAGA   3540                                                                 - - AGCAGGGGCA AGGCCAATGG ACATATCAAA TTTATCAAGA GCCATTTAAA AA -            #TCTGAAAA   3600                                                                 - - CAGGAAAATA TGCAAGAATG AAGGGTGCCC ACACTAATGA TGTGAAACAA TT -            #AACAGAGG   3660                                                                 - - CAGTACAAAA AATAGCCACA GAAAGCATAG TAATATGGGG AAAGACTCCT AA -            #ATTTAAAT   3720                                                                 - - TACCCATACA AAAGGAAACA TGGGAAGCAT GGTGGACAGA GTATTGGCAA GC -            #CACCTGGA   3780                                                                 - - TTCCTGAGTG GGAGTTTGTC AATACCCCTC CCTTAGTGAA GTTATGGTAC CA -            #GTTAGAGA   3840                                                                 - - AAGAACCCAT AATAGGAGCA GAAACTTTCT ATGTAGATGG GGCAGCCAAT AG -            #GGAAACTA   3900                                                                 - - AATTAGGAAA AGCAGGATAT GTAACTGACA GAGGAAGACA AAAAGTTGTC CC -            #CCTAACGG   3960                                                                 - - ACACAACAAA TCAGAAGACT GAGTTACAAG CAATTCATCT AGCTTTGCAG GA -            #TTCGGGAT   4020                                                                 - - TAGAAGTAAA CATAGTGACA GACTCACAAT ATGCATTGGG AATCATTCAA GC -            #ACAACCAG   4080                                                                 - - ATAAGAGTGA ATCAGAGTTA GTCAGTCAAA TAATAGAGCA GTTAATAAAA AA -            #GGAAAAAG   4140                                                                 - - TCTACCTGGC ATGGGTACCA GCACACAAAG GAATTGGAGG AAATGAACAA GT -            #AGATGGGT   4200                                                                 - - TGGTCAGTGC TGGAATCAGG AAAGTACTAT TTTTAGATGG AATAGATAAG GC -            #CCAAGAAG   4260                                                                 - - AACATGAGAA ATATCACAGT AATTGGAGAG CAATGGCTAG TGATTTTAAC CT -            #ACCACCTG   4320                                                                 - - TAGTAGCAAA AGAAATAGTA GCCAGCTGTG ATAAATGTCA GCTAAAAGGG GA -            #AGCCATGC   4380                                                                 - - ATGGACAAGT AGACTGTAGC CCAGGAATAT GGCAGCTAGA TTGTACACAT TT -            #AGAAGGAA   4440                                                                 - - AAGTTATCTT GGTAGCAGTT CATGTAGCCA GTGGATATAT AGAAGCAGAA GT -            #AATTCCAG   4500                                                                 - - CAGAGACAGG GCAAGAAACA GCATACTTCC TCTTAAAATT AGCAGGAAGA TG -            #GCCAGTAA   4560                                                                 - - AAACAGTACA TACAGACAAT GGCAGCAATT TCACCAGTAC TACAGTTAAG GC -            #CGCCTGTT   4620                                                                 - - GGTGGGCGGG GATCAAGCAG GAATTTGGCA TTCCCTACAA TCCCCAAAGT CA -            #AGGAGTAA   4680                                                                 - - TAGAATCTAT GAATAAAGAA TTAAAGAAAA TTATAGGACA GGTAAGAGAT CA -            #GGCTGAAC   4740                                                                 - - ATCTTAAGAC AGCAGTACAA ATGGCAGTAT TCATCCACAA TTTTAAAAGA AA -            #AGGGGGGA   4800                                                                 - - TTGGGGGGTA CAGTGCAGGG GAAAGAATAG TAGACATAAT AGCAACAGAC AT -            #ACAAACTA   4860                                                                 - - AAGAATTACA AAAACAAATT ACAAAAATTC AAAATTTTCG GGTTTATTAC AG -            #GGACAGCA   4920                                                                 - - GAGATCCAGT TTGGAAAGGA CCAGCAAAGC TCCTCTGGAA AGGTGAAGGG GC -            #AGTAGTAA   4980                                                                 - - TACAAGATAA TAGTGACATA AAAGTAGTGC CAAGAAGAAA AGCAAAGATC AT -            #CAGGGATT   5040                                                                 - - ATGGAAAACA GATGGCAGGT GATGATTGTG TGGCAAGTAG ACAGGATGAG GA -            #TTAACACA   5100                                                                 - - TGGAAAAGAT TAGTAAAACA CCATATGTAT ATTTCAAGGA AAGCTAAGGA CT -            #GGTTTTAT   5160                                                                 - - AGACATCACT ATGAAAGTAC TAATCCAAAA ATAAGTTCAG AAGTACACAT CC -            #CACTAGGG   5220                                                                 - - GATGCTAAAT TAGTAATAAC AACATATTGG GGTCTGCATA CAGGAGAAAG AG -            #ACTGGCAT   5280                                                                 - - TTGGGTCAGG GAGTCTCCAT AGAATGGAGG AAAAAGAGAT ATAGCACACA AG -            #TAGACCCT   5340                                                                 - - GACCTAGCAG ACCAACTAAT TCATCTGCAC TATTTTGATT GTTTTTCAGA AT -            #CTGCTATA   5400                                                                 - - AGAAATACCA TATTAGGACG TATAGTTAGT CCTAGGTGTG AATATCAAGC AG -            #GACATAAC   5460                                                                 - - AAGGTAGGAT CTCTACAGTA CTTGGCACTA GCAGCATTAA TAAAACCAAA AC -            #AGATAAAG   5520                                                                 - - CCACCTTTGC CTAGTGTTAG GAAACTGACA GAGGACAGAT GGAACAAGCC CC -            #AGAAGACC   5580                                                                 - - AAGGGCCACA GAGGGAGCCA TACAATGAAT GGACACTAGA GCTTTTAGAG GA -            #ACTTAAGA   5640                                                                 - - GTGAAGCTGT TAGACATTTT CCTAGGATAT GGCTCCATAA CTTAGGACAA CA -            #TATCTATG   5700                                                                 - - AAACTTACGG GGATACTTGG GCAGGAGTGG AAGCCATAAT AAGAATTCTG CA -            #ACAACTGC   5760                                                                 - - TGTTTATCCA TTTCAGAATT GGGTGTCGAC ATAGCAGAAT AGGCGTTACT CG -            #ACAGAGGA   5820                                                                 - - GAGCAAGAAA TGGAGCCAGT AGATCCTAGA CTAGAGCCCT GGAAGCATCC AG -            #GAAGTCAG   5880                                                                 - - CCTAAAACTG CTTGTACCAA TTGCTATTGT AAAAAGTGTT GCTTTCATTG CC -            #AAGTTTGT   5940                                                                 - - TTCATGACAA AAGCCTTAGG CATCTCCTAT GGCAGGAAGA AGCGGAGACA GC -            #GACGAAGA   6000                                                                 - - GCTCATCAGA ACAGTCAGAC TCATCAAGCT TCTCTATCAA AGCAGTAAGT AG -            #TACATGTA   6060                                                                 - - ATGCAACCTA TAATAGTAGC AATAGTAGCA TTAGTAGTAG CAATAATAAT AG -            #CAATAGTT   6120                                                                 - - GTGTGGTCCA TAGTAATCAT AGAATATAGG AAAATATTAA GACAAAGAAA AA -            #TAGACAGG   6180                                                                 - - TTAATTGATA GACTAATAGA AAGAGCAGAA GACAGTGGCA ATGAGAGTGA AG -            #GAGAAGTA   6240                                                                 - - TCAGCACTTG TGGAGATGGG GGTGGAAATG GGGCACCATG CTCCTTGGGA TA -            #TTGATGAT   6300                                                                 - - CTGTAGTGCT ACAGAAAAAT TGTGGGTCAC AGTCTATTAT GGGGTACCTG TG -            #TGGAAGGA   6360                                                                 - - AGCAACCACC ACTCTATTTT GTGCATCAGA TGCTAAAGCA TATGATACAG AG -            #GTACATAA   6420                                                                 - - TGTTTGGGCC ACACATGCCT GTGTACCCAC AGACCCCAAC CCACAAGAAG TA -            #GTATTGGT   6480                                                                 - - AAATGTGACA GAAAATTTTA ACATGTGGAA AAATGACATG GTAGAACAGA TG -            #CATGAGGA   6540                                                                 - - TATAATCAGT TTATGGGATC AAAGCCTAAA GCCATGTGTA AAATTAACCC CA -            #CTCTGTGT   6600                                                                 - - TAGTTTAAAG TGCACTGATT TGAAGAATGA TACTAATACC AATAGTAGTA GC -            #GGGAGAAT   6660                                                                 - - GATAATGGAG AAAGGAGAGA TAAAAAACTG CTCTTTCAAT ATCAGCACAA GC -            #ATAAGAGA   6720                                                                 - - TAAGGTGCAG AAAGAATATG CATTCTTTTA TAAACTTGAT ATAGTACCAA TA -            #GATAATAC   6780                                                                 - - CAGCTATAGG TTGATAAGTT GTAACACCTC AGTCATTACA CAGGCCTGTC CA -            #AAGGTATC   6840                                                                 - - CTTTGAGCCA ATTCCCATAC ATTATTGTGC CCCGGCTGGT TTTGCGATTC TA -            #AAATGTAA   6900                                                                 - - TAATAAGACG TTCAATGGAA CAGGACCATG TACAAATGTC AGCACAGTAC AA -            #TGTACACA   6960                                                                 - - TGGAATCAGG CCAGTAGTAT CAACTCAACT GCTGTTAAAT GGCAGTCTAG CA -            #GAAGAAGA   7020                                                                 - - TGTAGTAATT AGATCTGCCA ATTTCACAGA CAATGCTAAA ACCATAATAG TA -            #CAGCTGAA   7080                                                                 - - CACATCTGTA GAAATTAATT GTACAAGACC CAACAACAAT ACAAGAAAAA GT -            #ATCCGTAT   7140                                                                 - - CCAGAGGGGA CCAGGGAGAG CATTTGTTAC AATAGGAAAA ATAGGAAATA TG -            #AGACAAGC   7200                                                                 - - ACATTGTAAC ATTAGTAGAG CAAAATGGAA TGCCACTTTA AAACAGATAG CT -            #AGCAAATT   7260                                                                 - - AAGAGAACAA TTTGGAAATA ATAAAACAAT AATCTTTAAG CAATCCTCAG GA -            #GGGGACCC   7320                                                                 - - AGAAATTGTA ACGCACAGTT TTAATTGTGG AGGGGAATTT TTCTACTGTA AT -            #TCAACACA   7380                                                                 - - ACTGTTTAAT AGTACTTGGT TTAATAGTAC TTGGAGTACT GAAGGGTCAA AT -            #AACACTGA   7440                                                                 - - AGGAAGTGAC ACAATCACAC TCCCATGCAG AATAAAACAA TTTATAAACA TG -            #TGGCAGGA   7500                                                                 - - AGTAGGAAAA GCAATGTATG CCCCTCCCAT CAGTGGACAA ATTAGATGTT CA -            #TCAAATAT   7560                                                                 - - TACTGGGCTG CTATTAACAA GAGATGGTGG TAATAACAAC AATGGGTCCG AG -            #ATCTTCAG   7620                                                                 - - ACCTGGAGGA GGCGATATGA GGGACAATTG GAGAAGTGAA TTATATAAAT AT -            #AAAGTAGT   7680                                                                 - - AAAAATTGAA CCATTAGGAG TAGCACCCAC CAAGGCAAAG AGAAGAGTGG TG -            #CAGAGAGA   7740                                                                 - - AAAAAGAGCA GTGGGAATAG GAGCTTTGTT CCTTGGGTTC TTGGGAGCAG CA -            #GGAAGCAC   7800                                                                 - - TATGGGCGCA GCGTCAATGA CGCTGACGGT ACAGGCCAGA CAATTATTGT CT -            #GATATAGT   7860                                                                 - - GCAGCAGCAG AACAATTTGC TGAGGGCTAT TGAGGCGCAA CAGCATCTGT TG -            #CAACTCAC   7920                                                                 - - AGTCTGGGGC ATCAAACAGC TCCAGGCAAG AATCCTGGCT GTGGAAAGAT AC -            #CTAAAGGA   7980                                                                 - - TCAACAGCTC CTGGGGATTT GGGGTTGCTC TGGAAAACTC ATTTGCACCA CT -            #GCTGTGCC   8040                                                                 - - TTGGAATGCT AGTTGGAGTA ATAAATCTCT GGAACAGATT TGGAATAACA TG -            #ACCTGGAT   8100                                                                 - - GGAGTGGGAC AGAGAAATTA ACAATTACAC AAGCTTAATA CACTCCTTAA TT -            #GAAGAATC   8160                                                                 - - GCAAAACCAG CAAGAAAAGA ATGAACAAGA ATTATTGGAA TTAGATAAAT GG -            #GCAAGTTT   8220                                                                 - - GTGGAATTGG TTTAACATAA CAAATTGGCT GTGGTATATA AAATTATTCA TA -            #ATGATAGT   8280                                                                 - - AGGAGGCTTG GTAGGTTTAA GAATAGTTTT TGCTGTACTT TCTATAGTGA AT -            #AGAGTTAG   8340                                                                 - - GCAGGGATAT TCACCATTAT CGTTTCAGAC CCACCTCCCA ATCCCGAGGG GA -            #CCCGACAG   8400                                                                 - - GCCCGAAGGA ATAGAAGAAG AAGGTGGAGA GAGAGACAGA GACAGATCCA TT -            #CGATTAGT   8460                                                                 - - GAACGGATCC TTAGCACTTA TCTGGGACGA TCTGCGGAGC CTGTGCCTCT TC -            #AGCTACCA   8520                                                                 - - CCGCTTGAGA GACTTACTCT TGATTGTAAC GAGGATTGTG GAACTTCTGG GA -            #CGCAGGGG   8580                                                                 - - GTGGGAAGCC CTCAAATATT GGTGGAATCT CCTACAGTAT TGGAGTCAGG AA -            #CTAAAGAA   8640                                                                 - - TAGTGCTGTT AACTTGCTCA ATGCCACAGC CATAGCAGTA GCTGAGGGGA CA -            #GATAGGGT   8700                                                                 - - TATAGAAGTA TTACAAGCAG CTTATAGAGC TATTCGCCAC ATACCTAGAA GA -            #ATAAGACA   8760                                                                 - - GGGCTTGGAA AGGATTTTGC TATAAGATGG GTGGCAAGTG GTCAAAAAGT AG -            #TGTGATTG   8820                                                                 - - GATGGCCTGC TGTAAGGGAA AGAATGAGAC GAGCTGAGCA AGAAATGGCT AG -            #CAAAGGAG   8880                                                                 - - AAGAACTCTT CACTGGAGTT GTCCCAATTC TTGTTGAATT AGATGGTGAT GT -            #TAACGGCC   8940                                                                 - - ACAAGTTCTC TGTCAGTGGA GAGGGTGAAG GTGATGCAAC ATACGGAAAA CT -            #TACCCTGA   9000                                                                 - - AGTTCATCTG CACTACTGGC AAACTGCCTG TTCCATGGCC AACACTTGTC AC -            #TACTCTCT   9060                                                                 - - CTTATGGTGT TCAATGCTTT TCAAGATACC CGGATCATAT GAAACGGCAT GA -            #CTTTTTCA   9120                                                                 - - AGAGTGCCAT GCCCGAAGGT TATGTACAGG AAAGGACCAT CTTCTTCAAA GA -            #TGACGGCA   9180                                                                 - - ACTACAAGAC ACGTGCTGAA GTCAAGTTTG AAGGTGATAC CCTTGTTAAT AG -            #AATCGAGT   9240                                                                 - - TAAAAGGTAT TGACTTCAAG GAAGATGGCA ACATTCTGGG ACACAAATTG GA -            #ATACAACT   9300                                                                 - - ATAACTCACA CAATGTATAC ATCATGGCAG ACAAACAAAA GAATGGAATC AA -            #AGTGAACT   9360                                                                 - - TCAAGACCCG CCACAACATT GAAGATGGAA GCGTTCAACT AGCAGACCAT TA -            #TCAACAAA   9420                                                                 - - ATACTCCAAT TGGCGATGGC CCTGTCCTTT TACCAGACAA CCATTACCTG TC -            #CACACAAT   9480                                                                 - - CTGCCCTTTC GAAAGATCCC AACGAAAAGA GAGACCACAT GGTCCTTCTT GA -            #GTTTGTAA   9540                                                                 - - CAGCTGCTGG GATTACACAT GGCATGGATG AACTGTACAA CGGACTCGAG AC -            #CTAGAAAA   9600                                                                 - - ACATGGAGCA ATCACAAGTA GCAATACAGC AGCTAACAAT GCTGCTTGTG CC -            #TGGCTAGA   9660                                                                 - - AGCACAAGAG GAGGAAGAGG TGGGTTTTCC AGTCACACCT CAGGTACCTT TA -            #AGACCAAT   9720                                                                 - - GACTTACAAG GCAGCTGTAG ATCTTAGCCA CTTTTTAAAA GAAAAGGGGG GA -            #CTGGAAGG   9780                                                                 - - GCTAATTCAC TCCCAAAGAA GACAAGATAT CCTTGATCTG TGGATCTACC AC -            #ACACAAGG   9840                                                                 - - CTACTTCCCT GATTGGCAGA ACTACACACC AGGGCCAGGG GTCAGATATC CA -            #CTGACCTT   9900                                                                 - - TGGATGGTGC TACAAGCTAG TACCAGTTGA GCCAGATAAG GTAGAAGAGG CC -            #AATAAAGG   9960                                                                 - - AGAGAACACC AGCTTGTTAC ACCCTGTGAG CCTGCATGGA ATGGATGACC CT -            #GAGAGAGA  10020                                                                 - - AGTGTTAGAG TGGAGGTTTG ACAGCCGCCT AGCATTTCAT CACGTGGCCC GA -            #GAGCTGCA  10080                                                                 - - TCCGGAGTAC TTCAAGAACT GCTGACATCG AGCTTGCTAC AAGGGACTTT CC -            #GCTGGGGA  10140                                                                 - - CTTTCCAGGG AGGCGTGGCC TGGGCGGGAC TGGGGAGTGG CGAGCCCTCA GA -            #TGCTGCAT  10200                                                                 - - ATAAGCAGCT GCTTTTTGCC TGTACTGGGT CTCTCTGGTT AGACCAGATC TG -            #AGCCTGGG  10260                                                                 - - AGCTCTCTGG CTAACTAGGG AACCCACTGC TTAAGCCTCA ATAAAGCTTG CC -            #TTGAGTGC  10320                                                                 - - TTCAAGTAGT GTGTGCCCGT CTGTTGTGTG ACTCTGGTAA CTAGAGATCC CT -            #CAGACCCT  10380                                                                 - - TTTAGTCAGT GTGGAAAATC TCTAGCACCC CCCAGGAGGT AGAGGTTGCA GT -            #GAGCCAAG  10440                                                                 - - ATCGCGCCAC TGCATTCCAG CCTGGGCAAG AAAACAAGAC TGTCTAAAAT AA -            #TAATAATA  10500                                                                 - - AGTTAAGGGT ATTAAATATA TTTATACATG GAGGTCATAA AAATATATAT AT -            #TTGGGCTG  10560                                                                 - - GGCGCAGTGG CTCACACCTG CGCCCGGCCC TTTGGGAGGC CGAGGCAGGT GG -            #ATCACCTG  10620                                                                 - - AGTTTGGGAG TTCCAGACCA GCCTGACCAA CATGGAGAAA CCCCTTCTCT GT -            #GTATTTTT  10680                                                                 - - AGTAGATTTT ATTTTATGTG TATTTTATTC ACAGGTATTT CTGGAAAACT GA -            #AACTGTTT  10740                                                                 - - TTCCTCTACT CTGATACCAC AAGAATCATC AGCACAGAGG AAGACTTCTG TG -            #ATCAAATG  10800                                                                 - - TGGTGGGAGA GGGAGGTTTT CACCAGCACA TGAGCAGTCA GTTCTGCCGC AG -            #ACTCGGCG  10860                                                                 - - GGTGTCCTTC GGTTCAGTTC CAACACCGCC TGCCTGGAGA GAGGTCAGAC CA -            #CAGGGTGA  10920                                                                 - - GGGCTCAGTC CCCAAGACAT AAACACCCAA GACATAAACA CCCAACAGGT CC -            #ACCCCGCC  10980                                                                 - - TGCTGCCCAG GCAGAGCCGA TTCACCAAGA CGGGAATTAG GATAGAGAAA GA -            #GTAAGTCA  11040                                                                 - - CACAGAGCCG GCTGTGCGGG AGAACGGAGT TCTATTATGA CTCAAATCAG TC -            #TCCCCAAG  11100                                                                 - - CATTCGGGGA TCAGAGTTTT TAAGGATAAC TTAGTGTGTA GGGGGCCAGT GA -            #GTTGGAGA  11160                                                                 - - TGAAAGCGTA GGGAGTCGAA GGTGTCCTTT TGCGCCGAGT CAGTTCCTGG GT -            #GGGGGCCA  11220                                                                 - - CAAGATCGGA TGAGCCAGTT TATCAATCCG GGGGTGCCAG CTGATCCATG GA -            #GTGCAGGG  11280                                                                 - - TCTGCAAAAT ATCTCAAGCA CTGATTGATC TTAGGTTTTA CAATAGTGAT GT -            #TACCCCAG  11340                                                                 - - GAACAATTTG GGGAAGGTCA GAATCTTGTA GCCTGTAGCT GCATGACTCC TA -            #AACCATAA  11400                                                                 - - TTTCTTTTTT GTTTTTTTTT TTTTATTTTT GAGACAGGGT CTCACTCTGT CA -            #CCTAGGCT  11460                                                                 - - GGAGTGCAGT GGTGCAATCA CAGCTCACTG CAGCCTCAAC GTCGTAAGCT CA -            #AGCGATCC  11520                                                                 - - TCCCACCTCA GCCTGCCTGG TAGCTGAGAC TACAAGCGAC GCCCCAGTTA AT -            #TTTTGTAT  11580                                                                 - - TTTTGGTAGA GGCAGCGTTT TGCCGTGTGG CCCTGGCTGG TCTCGAACTC CT -            #GGGCTCAA  11640                                                                 - - GTGATCCAGC CTCAGCCTCC CAAAGTGCTG GGACAACCGG GGCCAGTCAC TG -            #CACCTGGC  11700                                                                 - - CCTAAACCAT AATTTCTAAT CTTTTGGCTA ATTTGTTAGT CCTACAAAGG CA -            #GTCTAGTC  11760                                                                 - - CCCAGGCAAA AAGGGGGTTT GTTTCGGGAA AGGGCTGTTA CTGTCTTTGT TT -            #CAAACTAT  11820                                                                 - - AAACTAAGTT CCTCCTAAAC TTAGTTCGGC CTACACCCAG GAATGAACAA GG -            #AGAGCTTG  11880                                                                 - - GAGGTTAGAA GCACGATGGA ATTGGTTAGG TCAGATCTCT TTCACTGTCT GA -            #GTTATAAT  11940                                                                 - - TTTGCAATGG TGGTTCAAAG ACTGCCCGCT TCTGACACCA GTCGCTGCAT TA -            #ATGAATCG  12000                                                                 - - GCCAACGCGC GGGGAGAGGC GGTTTGCGTA TTGGCGCTCT TCCGCTTCCT CG -            #CTCACTGA  12060                                                                 - - CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AG -            #GCGGTAAT  12120                                                                 - - ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AA -            #GGCCAGCA  12180                                                                 - - AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TC -            #CGCCCCCC  12240                                                                 - - TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CA -            #GGACTATA  12300                                                                 - - AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CG -            #ACCCTGCC  12360                                                                 - - GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CT -            #CAATGCTC  12420                                                                 - - ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GT -            #GTGCACGA  12480                                                                 - - ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AG -            #TCCAACCC  12540                                                                 - - GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GC -            #AGAGCGAG  12600                                                                 - - GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT AC -            #ACTAGAAG  12660                                                                 - - GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GA -            #GTTGGTAG  12720                                                                 - - CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GC -            #AAGCAGCA  12780                                                                 - - GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CG -            #GGGTCTGA  12840                                                                 - - CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CA -            #AAAAGGAT  12900                                                                 - - CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GT -            #ATATATGA  12960                                                                 - - GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CA -            #GCGATCTG  13020                                                                 - - TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CG -            #ATACGGGA  13080                                                                 - - GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CA -            #CCGGCTCC  13140                                                                 - - AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GT -            #CCTGCAAC  13200                                                                 - - TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GT -            #AGTTCGCC  13260                                                                 - - AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CA -            #CGCTCGTC  13320                                                                 - - GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CA -            #TGATCCCC  13380                                                                 - - CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GA -            #AGTAAGTT  13440                                                                 - - GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CT -            #GTCATGCC  13500                                                                 - - ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GA -            #GAATAGTG  13560                                                                 - - TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CG -            #CCACATAG  13620                                                                 - - CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TC -            #TCAAGGAT  13680                                                                 - - CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GA -            #TCTTCAGC  13740                                                                 - - ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA AT -            #GCCGCAAA  13800                                                                 - - AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TT -            #CAATATTA  13860                                                                 - - TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GT -            #ATTTAGAA  13920                                                                 - - AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG AC -            #GTCTAAGA  13980                                                                 - - AACCATTATT ATCATGACAT TAACCTATAA AAATAGGCGT ATCACGAGGC CC -            #TTTCGTCT  14040                                                                 - - TCAAGAACTG CCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC AT -            #GCAGCTCC  14100                                                                 - - CGGAGACGGT CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CG -            #TCAGGGCG  14160                                                                 - - CGTCAGCGGG TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AG -            #CGATAGCG  14220                                                                 - - GAGTGTACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT ACTGAGAGTG CA -            #CCATATGC  14280                                                                 - - GGTGTGAAAT ACCGCACAGA TGCGTAAGGA GAAAATACCG CATCAGGCGC CA -            #TTCGCCAT  14340                                                                 - - TCAGGCTGCG CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TT -            #ACGCCAGC  14400                                                                 - - GCGGGGAGGC AGAGATTGCA GTAAGCTGAG ATCGCAGCAC TGCACTCCAG CC -            #TGGGCGAC  14460                                                                 - - AGAGTAAGAC TCTGTCTCAA AAATAAAATA AATAAATCAA TCAGATATTC CA -            #ATCTTTTC  14520                                                                 - - CTTTATTTAT TTATTTATTT TCTATTTTGG AAACACAGTC CTTCCTTATT CC -            #AGAATTAC  14580                                                                 - - ACATATATTC TATTTTTCTT TATATGCTCC AGTTTTTTTT AGACCTTCAC CT -            #GAAATGTG  14640                                                                 - - TGTATACAAA ATCTAGGCCA GTCCAGCAGA GCCTAAAGGT AAAAAATAAA AT -            #AATAAAAA  14700                                                                 - - ATAAATAAAA TCTAGCTCAC TCCTTCACAT CAAAATGGAG ATACAGCTGT TA -            #GCATTAAA  14760                                                                 - - TACCAAATAA CCCATCTTGT CCTCAATAAT TTTAAGCGCC TCTCTCCACC AC -            #ATCTAACT  14820                                                                 - - CCTGTCAAAG GCATGTGCCC CTTCCGGGCG CTCTGCTGTG CTGCCAACCA AC -            #TGGCATGT  14880                                                                 - - GGACTCTGCA GGGTCCCTAA CTGCCAAGCC CCACAGTGTG CCCTGAGGCT GC -            #CCCTTCCT  14940                                                                 - - TCTAGCGGCT GCCCCCACTC GGCTTTGCTT TCCCTAGTTT CAGTTACTTG CG -            #TTCAGCCA  15000                                                                 - - AGGTCTGAAA CTAGGTGCGC ACAGAGCGGT AAGACTGCGA GAGAAAGAGA CC -            #AGCTTTAC  15060                                                                 - - AGGGGGTTTA TCACAGTGCA CCCTGACAGT CGTCAGCCTC ACAGGGGGTT TA -            #TCACATTG  15120                                                                 - - CACCCTGACA GTCGTCAGCC TCACAGGGGG TTTATCACAG TGCACCCTTA CA -            #ATCATTCC  15180                                                                 - - ATTTGATTCA CAATTTTTTT AGTCTCTACT GTGCCTAACT TGTAAGTTAA AT -            #TTGATCAG  15240                                                                 - - AGGTGTGTTC CCAGAGGGGA AAACAGTATA TACAGGGTTC AGTACTATCG CA -            #TTTCAGGC  15300                                                                 - - CTCCACCTGG GTCTTGGAAT GTGTCCCCCG AGGGGTGATG ACTACCTCAG TT -            #GGATCTCC  15360                                                                 - - ACAGGTCACA GTGACACAAG ATAACCAAGA CACCTCCCAA GGCTACCACA AT -            #GGGCCGCC  15420                                                                 - - CTCCACGTGC ACATGGCCGG AGGAACTGCC ATGTCGGAGG TGCAAGCACA CC -            #TGCGCATC  15480                                                                 - - AGAGTCCTTG GTGTGGAGGG AGGGACCAGC GCAGCTTCCA GCCATCCACC TG -            #ATGAACAG  15540                                                                 - - AACCTAGGGA AAGCCCCAGT TCTACTTACA CCAGGAAAGG C    - #                      - #15581                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..74                                                           (D) OTHER INFORMATION: - #/note= "primer #17982"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - GGGGCGTACG GAGCGCTCCG AATTCGGTAC CGTTTAAACG GGCCCTCTCG AG -             #TCCGTTGT     60                                                                 - - ACAGTTCATC CATG              - #                  - #                      - #     74                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: -                                                               (B) LOCATION: 1..66                                                           (D) OTHER INFORMATION: - #/note= "primer #17983"                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - GGGGGAATTC GCGCGCGTAC GTAAGCGCTA GCTGAGCAAG AAATGGCTAG CA -             #AAGGAGAA     60                                                                 - - GAACTC                 - #                  - #                  -     #           66                                                                __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid that encodes anengineered Aequorea victoria fluorescent protein, wherein the proteinencoded by the isolated nucleic acid is selected from the group thatconsists of proteins wherein one or more amino acid residues of aprotein having an amino acid sequence set forth in SEQ ID NO:2 aresubstituted to produce:a. a singly-substituted protein that has leucineat amino acid position 65, and wherein said protein has a cellularfluorescence that is at least about ten times greater than the cellularfluorescence of a protein having the amino acid sequence set forth inSEQ ID NO:2; b. a protein that has leucine at amino acid position 65 andthreonine at position 168, and wherein said protein has a cellularfluorescence that is at least about ten times greater than a proteinhaving the amino acid sequence set forth in SEQ ID NO:2; c. a proteinthat has leucine at amino acid position 65, threonine at position 168,and cysteine at position 66, wherein said protein has a cellularfluorescence that is at least about ten times greater than the cellularfluorescence of a protein having the amino acid sequence set forth inSEQ ID NO:2; d. a blue fluorescent protein (BFP) that has histidine atamino acid position 67, leucine at position 65 and has a cellularfluorescence that is at least five times greater than that of BFP(Tyr₆₇→His); e. a blue fluorescent protein that has histidine at amino acidposition 67, alanine at amino acid position 164 and has a cellularfluorescence that is at least five times greater than that of BFP(Tyr₆₇→His); f. a blue fluorescent protein that has histidine at amino acidposition 67, leucine at amino acid position 65, alanine at amino acidposition 164 and has a cellular fluorescence that is at least five timesgreater than that of BFP(Tyr₆₇ →His).
 2. An isolated nucleic acid ofclaim 1, which encodes an engineered Aequorea victoria green fluorescentprotein (GFP) having a cellular fluorescence that is at least about tentimes greater than that of the protein having the amino acid sequenceset forth in SEQ ID NO:2, wherein the engineered GFP has a leucine atamino acid position
 65. 3. An isolated nucleic acid according to claim 1which encodes an engineered Aequorea victoria green fluorescent protein(GFP) having a cellular fluorescence that is at least about ten timesgreater than that of the protein having the amino acid sequence setforth in SEQ ID NO:2, wherein the engineered GFP has a leucine at aminoacid position 65 and a threonine at amino acid position
 168. 4. Anisolated nucleic acid according to claim 3, wherein the nucleic acidfurther encodes a cysteine at amino acid position
 66. 5. An isolatednucleic acid of claim 1 that encodes an engineered blue fluorescentprotein BFP that has histidine at amino acid position 67 and leucine atposition 65, and has a cellular fluorescence that is at least five timesgreater than that of BFP(Tyr₆₇ →His).
 6. An isolated nucleic acid ofclaim 1 that encodes an engineered blue fluorescent protein (BFP) thathas histidine at amino acid position 67 and alanine at amino acidposition 164, and has a cellular fluorescence that is at least fivetimes greater than that of BFP(Tyr₆₇ →His).
 7. An isolated nucleic acidaccording to claim 6, wherein the nucleic acid further encodes leucineat amino acid position
 65. 8. A transformed cell that expresses aprotein encoded by a nucleic acid of claim
 1. 9. A vector comprising anucleic acid of claim
 1. 10. A transformed cell comprising a vector ofclaim
 9. 11. A transformed cell that expresses a protein encoded by thenucleic acid of claim 1 fused to a protein encoded by a second nucleicacid of interest.
 12. A method of detecting and optionally isolating anengineered cell that contains a first selected nucleic acid and a secondselected nucleic acid, comprising:a) stably introducing into a host cellin a population of host cells a vector that contains a first nucleicacid selected from the group consisting of nucleic acids that encodeSG11, SG12, SG25, SB42, SB49, and SB50, and a second nucleic acid whichencodes a selected protein or a nucleic acid regulatory sequence, and b)detecting cells in the population of host cells that express SG11, SG12,SG25, SB42, SB49, or SB50, and c) optionally sorting cells that expressSG11, SG12, SG25, SB42, SB49, or SB50 with a fluorescence-activated cellsorter to isolate individual cells that express said fluorescentprotein.
 13. A nucleic acid construct wherein a coding sequence selectedfrom the group consisting of nucleic acids that encode SG11, SG12, SG25,SB42, SB49, and SB50 is operably linked to a regulatory sequence of aselected gene.
 14. A nucleic acid construct wherein a first codingsequence that encodes a selected polypeptide is fused using geneticengineering to a second coding sequence selected from the groupconsisting of nucleic acids that encode SG11, SG12, SG25, SB42, SB49,and SB50, such that expression of the fused sequence yields afluorescent hybrid protein in which the polypeptide encoded by the firstcoding sequence is fused to the polypeptide encoded by the second codingsequence.
 15. A method of detecting and characterizing regulatory andcoding sequence elements that regulate subcellular expression andtargeting of proteins, comprising:a) expressing in an engineered cell,in the presence and absence of selected culture conditions andcomponents, a nucleic acid wherein a first nucleic acid selected fromthe group consisting nucleic acids that encode SG11, SG12, SG25, SB42,SB49, and SB50 is operably linked to a second nucleic acid derived froma selected gene; b) detecting the presence and subcellular localizationof fluorescent signal.
 16. A method according to claim 12, wherein saidnucleic acid that encodes SG11 has the nucleic acid sequence set forthin SEQ ID NO:15, said nucleic acid that encodes SG12 has the nucleicacid sequence set forth in SEQ ID NO:16, said nucleic acid that encodesSG25 has the nucleic acid sequence set forth in SEQ ID NO: 17, saidnucleic acid that encodes SB42 has the nucleic acid sequence set forthin SEQ ID NO: 19, said nucleic acid that encodes SB49 has the nucleicacid sequence set forth in SEQ ID NO:21, and said nucleic acid thatencodes SB50 has the nucleic acid sequence set forth in SEQ ID NO:24.17. A nucleic acid construct according to claim 13, wherein said nucleicacid that encodes SG11 has the nucleic acid sequence set forth in SEQ IDNO:15, said nucleic acid that encodes SG12 has the nucleic acid sequenceset forth in SEQ ID NO: 16, said nucleic acid that encodes SG25 has thenucleic acid sequence set forth in SEQ ID NO:17, said nucleic acid thatencodes SB42 has the nucleic acid sequence set forth in SEQ ID NO: 19,said nucleic acid that encodes SB49 has the nucleic acid sequence setforth in SEQ ID NO:21, and said nucleic acid that encodes SB50 has thenucleic acid sequence set forth in SEQ ID NO:24.
 18. A nucleic acidconstruct according to claim 14, wherein said nucleic acid that encodesSG11 has the nucleic acid sequence set forth in SEQ ID NO:15, saidnucleic acid that encodes SG12 has the nucleic acid sequence set forthin SEQ ID NO: 16, said nucleic acid that encodes SG25 has the nucleicacid sequence set forth in SEQ ID NO: 17, said nucleic acid that encodesSB42 has the nucleic acid sequence set forth in SEQ ID NO: 19, saidnucleic acid that encodes SB49 has the nucleic acid sequence set forthin SEQ ID NO:21, and said nucleic acid that encodes SB50 has the nucleicacid sequence set forth in SEQ ID NO:24.
 19. A method according to claim15, wherein said nucleic acid that encodes SG11 has the nucleic acidsequence set forth in SEQ ID NO:15, said nucleic acid that encodes SG12has the nucleic acid sequence set forth in SEQ ID NO:16, said nucleicacid that encodes SG25 has the nucleic acid sequence set forth in SEQ IDNO:17, said nucleic acid that encodes SB42 has the nucleic acid sequenceset forth in SEQ ID NO:19, said nucleic acid that encodes SB49 has thenucleic acid sequence set forth in SEQ ID NO:21, and said nucleic acidthat encodes SB50 has the nucleic acid sequence set forth in SEQ IDNO:24.
 20. An isolated nucleic acid of claim 1, which encodes anengineered Aequorea victoria green fluorescent protein (GFP) having acellular fluorescence that is at least about twenty times greater thanthat of the protein having the amino acid sequence set forth in SEQ IDNO:2.
 21. An isolated nucleic acid of claim 1 which encodes anengineered GFP having a cellular fluorescence that is at least about tentimes greater than that of the protein having the amino acid sequenceset forth in SEQ ID NO:2 wherein said protein is selected from the groupconsisting of: SG12 (F65L), SG11 (F65L, I168T, K239N), SG25 (F65L, S66C,I168T, K239N), SG30 (F47L, F65L, I168T, K239N), SG32 (F65L, F72L, I168T,K239N), SG43 (F65L, I168T, Y201L, K239N), SG46 (F65L, V164A, I168T,K239N), SG72 (F65L, S66C, V164A, I168T, K239N), SG91 (F65L, S66C, F100L,I168T, K239N), SG94 (F65L, S66C, Y107L, I168T, K239N), SG95 (F65L, S66C,F115L, I168T, K239N), SG96 (F65L, S66C, F131L, I168T, K239N), SG98(F65L, S66C, Y146L, I168T, K239N), SG100 (F65L, S66C, Y152L, I168T,K239N), SG101 (F65L, S66C, I168T Y183L, K239N), SG102 (65L, S66C, I168T,F224L, K239N), SG103 (F65L, S66C, I168T, Y238L, K239N), and SG106 (F65L,S66T, V164A, I168T, K239N).